Compositions and methods for delivery of biomacromolecule agents

ABSTRACT

The present invention relates to nanoparticles associated with (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) biomacromolecule agents configured for treating, preventing or ameliorating various types of disorders, and methods of synthesizing the same. In particular, the present invention is directed to compositions comprising nanoparticles (e.g., synthetic high density lipoprotein (sHDL)) associated with (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) biomacromolecule agents (e.g., nucleic acid, peptides, glycolipids, etc.), methods for synthesizing such nanoparticles, as well as systems and methods utilizing such nanoparticles (e.g., in diagnostic and/or therapeutic settings).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI097291 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to nanoparticles associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed)biomacromolecule agents configured for treating, preventing orameliorating various types of disorders, and methods of synthesizing thesame. In particular, the present invention is directed to compositionscomprising nanoparticles (e.g., synthetic high density lipoprotein(sHDL)) associated with (e.g., complexed, conjugated, encapsulated,absorbed, adsorbed, admixed) biomacromolecule agents (e.g., nucleicacid, peptides, glycolipids, etc.), methods for synthesizing suchnanoparticles, as well as systems and methods utilizing suchnanoparticles (e.g., in diagnostic and/or therapeutic settings).

BACKGROUND OF THE INVENTION

Peptide and nucleic acid based drugs have tremendous potential as thenext generation therapeutics. Despite their huge potential, theirclinical translation has been challenging, partially due to lack of drugdelivery platforms that can efficiently deliver the drugs to the site ofaction while protecting the cargo materials against enzymaticdegradation in vivo. One prime example is in the area of cancervaccines; numerous clinical trials have been performed using definedtumor associated antigen peptides, but they have failed to demonstrateclinical efficacy because soluble peptides do not sufficiently reach thesite of action (e.g., lymphoid tissues) and fail to generate strongimmune responses.

Improved compositions and techniques for stable and targeted delivery(e.g., in vitro or in vivo) of biomacromolcules (e.g., peptides, nucleicacids, glycolipids) are needed.

SUMMARY

Despite the tremendous potential of peptide-based cancer vaccines, theirefficacy has been limited in humans. Recent innovations in tumor exomesequencing have signaled the new era of “personalized” immunotherapywith patient-specific neo-antigens (see, e.g., Yadav, M. et al. Nature515, 572-576 (2014); Kreiter, S. et al. Nature 520, 692-696 (2015);Schumacher, T. N. & Schreiber, R. D. Science 348, 69-74 (2015)), but ageneral methodology for stimulating strong CD8α+ cytotoxic T lymphocyte(CTL) responses remains lacking.

Experiments conducted during the course of developing embodiments forthe present invention demonstrated that preformed high densitylipoprotein-mimicking nanodiscs can be readily coupled with antigen (Ag)peptides and adjuvants, producing stable, ultrasmall nanoparticles thatmarkedly improve Ag/adjuvant co-delivery to lymphoid organs and achievedsustained Ag presentation on dendritic cells. Strikingly, it was shownthat these nanodiscs elicited up to 41-fold greater frequency of CTLsthan soluble vaccines and even 9-fold greater than perhaps the strongestadjuvant in clinical trials (i.e. CpG in Montanide) (see, e.g., Speiser,D. E. et al. J. Clin. Invest. 115, 739-746 (2005); Fourcade, J. et al.J. Immunother. 31, 781-791 (2008)). Moreover, it was shown that thenanodisc platform can be easily adapted to neoantigens, generatingpotent anti-tumor immunity. Such results represent a new powerfulapproach for cancer immunotherapy and more broadly, suggest a generalstrategy for personalized nanomedicine.

Such results have significant clinical importance, as these nanodiscs,with an established manufacturing procedure and excellent safetyprofiles in humans, can drastically improve co-delivery of antigens andadjuvants to LNs, sustain antigen presentation on DCs, and drive T-cellresponses with potent anti-tumor efficacy. As the majority of tumormutations are unique to each patient, cancer vaccines would require apersonalized approach (see, e.g., Yadav, M. et al. Nature 515, 572-576(2014); Kreiter, S. et al. Nature 520, 692-696 (2015); Schumacher, T. N.& Schreiber, R. D. Science 348, 69-74 (2015)). Coupled with the recenttechnical innovations in neo-antigen screening, this approach providespowerful yet facile strategies for producing cancer vaccines designedfor each patient. Furthermore, this platform technology is generallyapplicable for personalized therapeutics with a wide range of bioactivemolecules and imaging agents.

Accordingly, in certain embodiments, the present invention providesmethods for making a personalized neoplasia vaccine for a subjectdiagnosed as having a neoplasia. The present invention is not limited toparticular methods for making a personalized neoplasia vaccine for asubject diagnosed as having a neoplasia. In some embodiments, suchmethods comprise obtaining a biological sample of the neoplasia from thesubject; identifying a plurality of mutations in the neoplasia;analyzing the plurality of mutations to identify one or moreneo-antigenic mutations predicted to encode neo-antigenic peptides, theneo-antigenic mutations selected from the group consisting of missensemutations, neoORF mutations, and any combination thereof; and producinga personalized neoplasia vaccine, wherein the personalized neoplasiavaccine comprises a microparticle or nanoparticle associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) one ormore neo-antigenic peptides specific for the analyzed and identifiedneo-antigenic mutations predicted to encode neo-antigenic peptides. Insome embodiments, the nanoparticle is further associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) withan adjuvant. In some embodiments, the identifying further comprisessequencing the genome, transcriptome, or proteome of the neoplasia.

In some embodiments, the size of the microparticle is between 0.5microns to 100 microns.

In some embodiments, the one or more neo-antigenic peptides range fromabout 5 to about 50 amino acids in length. In some embodiments, the oneor more neo-antigenic mutations peptides range from about 15 to about 35amino acids in length. In some embodiments, the one or moreneo-antigenic peptides range from about 18 to about 30 amino acids inlength. In some embodiments, the one or more neo-antigenic peptidesrange from about 6 to about 15 amino acids in length.

In some embodiments, the adjuvant is selected from the group consistingof CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts (for example,aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870,893,CpG7909, CyaA, dSLIM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L),IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGA microparticles,imiquimod, resiquimod, gardiquimod, 3M-052, SRL172, Virosomes and otherVirus-like particles, YF-17D, VEGF trap, beta-glucan, Pam3Cys, Aquila'sQS21 stimulon, vadimezan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyllipid adjuvant (GLA), GLA-SE, CD1d ligands (such as C20:2, OCH, AH04-2,α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide,α-fructosylceramide, β-galatosylceramide, β-mannosylceramide), STINGagonists (e.g cyclic dinucleotides, including Cyclic[G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p], Cyclic[G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), bacterialtoxins (such as CT, and LT). In some embodiments, the adjuvant is anyderivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof.

The methods are not limited to a particular nanoparticle. In someembodiments, the average size of the nanoparticle is between 6 to 500nm. In some embodiments, the nanoparticle is a sHDL nanoparticle. Insome embodiments, the sHDL nanoparticle comprises a mixture of at leastone phospholipid and at least one HDL apolipoprotein or apolipoproteinmimetic. In some embodiments, the average size of the nanoparticle isbetween 6 to 500 nm. In some embodiments, the average particle size ofthe sHDL nanoparticle is between 6-70 nm.

In some embodiments, the phospholipid is selected from the groupconsisting of dipalmitoylphosphatidylcholine (DPPC),dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof.

In some embodiments, the HDL apolipoprotein component is selected fromthe group consisting of apolipoprotein A-I (apo A-I), apolipoproteinA-II (apo A-II), apolipoprotein A-II xxx (apo A-II-xxx), apolipoproteinA4 (apo A4), apolipoprotein Cs (apo Cs), apolipoprotein E (apo E),apolipoprotein A-I milano (apo A-I-milano), apolipoprotein A-I paris(apo A-I-paris), apolipoprotein M (apo M), an HDL apolipoproteinmimetic, preproapoliprotein, preproApoA-I, proApoA I, preproApoA-II,proApoA II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE,preproApoA I_(Milano), proApoA-I_(Milano), preproApoA-I_(Paris),proApoA-I_(Paris), and mixtures thereof.

In some embodiments, the ApoA-I mimetic is described by any of

SEQ ID NOs: 1-336 and (SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF,(SEQ ID NO: 342) LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343)PVTOEFWDNLEKETEGLROEMS, (SEQ ID NO: 344) KDLEEVKAKVQ, (SEQ ID NO: 345)KDLEEVKAKVO, (SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS, (SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.

In certain embodiments, the present invention provides methods fortreating a subject diagnosed as having a neoplasia with a personalizedneoplasia vaccine. The present invention is not limited to particularmethods for treating a subject diagnosed as having a neoplasia with apersonalized neoplasia vaccine. In some embodiments, such methodscomprise obtaining a biological sample of the neoplasia from thesubject; identifying one or more mutations in the neoplasia; analyzingthe plurality of mutations to identify one or more neo-antigenicmutations predicted to encode expressed neo-antigenic peptides, theneo-antigenic mutations selected from the group consisting of missensemutations, neoORF mutations, and any combination thereof; producing apersonalized neoplasia vaccine, wherein the personalized neoplasiavaccine comprises a microparticle or nanoparticle associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) one ormore neo-antigenic peptides specific for the analyzed and identifiedneo-antigenic mutations predicted to encode neo-antigenic peptides; andadministering the personalized neoplasia vaccine to the subject, therebytreating the neoplasia. In some embodiments, the personalized neoplasiavaccine is coadministered with an adjuvant. In some embodiments, thenanoparticle is further associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) an adjuvant. In someembodiments, the identifying further comprises sequencing the genome,transcriptome, or proteome of the neoplasia.

In some embodiments, the one or more neo-antigenic peptides range fromabout 5 to about 50 amino acids in length. In some embodiments, the oneor more neo-antigenic mutations peptides range from about 15 to about 35amino acids in length. In some embodiments, the one or moreneo-antigenic peptides range from about 18 to about 30 amino acids inlength. In some embodiments, the one or more neo-antigenic peptidesrange from about 6 to about 15 amino acids in length.

In some embodiments, the adjuvant is selected from the group consistingof CPG, polylC, poly-ICLC, 1018 ISS, aluminum salts (for example,aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870,893,CpG7909, CyaA, dSLIM, Cytokines (such as GM-CSF, IL-2, IFN-a, Fit-3L),IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGA microparticles,imiquimod, resiquimod, gardiquimod, 3M-052, SRL172, Virosomes and otherVirus-like particles, YF-17D, VEGF trap, beta-glucan, Pam3Cys, Aquila'sQS21 stimulon, vadimezan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyllipid adjuvant (GLA), GLA-SE, CD1d ligands (such as C20:2, OCH, AH04-2,α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide,α-fructosylceramide, β-galatosylceramide, β-mannosylceramide), STINGagonists (e.g. cyclic dinucleotides, including Cyclic[G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p], Cyclic[G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, 1C31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), bacterialtoxins (such as CT, and LT). In some embodiments, the adjuvant is anyderivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof.

The methods are not limited to a particular nanoparticle. In someembodiments, the average size of the nanoparticle is between 6 to 500nm. In some embodiments, the nanoparticle is a sHDL nanoparticle. Insome embodiments, the sHDL nanoparticle comprises a mixture of at leastone phospholipid and at least one HDL apolipoprotein or apolipoproteinmimetic. In some embodiments, the average particle size of the sHDLnanoparticle is between 6-70 nm.

In some embodiments, the phospholipid is selected from the groupconsisting of dipalmitoylphosphatidylcholine (DPPC),dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof.

In some embodiments, the HDL apolipoprotein component is selected fromthe group consisting of apolipoprotein A-I (apo A-I), apolipoproteinA-II (apo A-II), apolipoprotein A-II xxx (apo A-II-xxx), apolipoproteinA4 (apo A4), apolipoprotein Cs (apo Cs), apolipoprotein E (apo E),apolipoprotein A-I milano (apo A-I-milano), apolipoprotein A-I paris(apo A-I-paris), apolipoprotein M (apo M), an HDL apolipoproteinmimetic, preproapoliprotein, preproApoA-I, proApoA I, preproApoA-II,proApoA II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE,preproApoA I_(Milano), proApoA-I_(Milano), preproApoA-I_(Paris),proApoA-I_(Paris), and mixtures thereof.

In some embodiments, the ApoA-I mimetic is described by any of

SEQ ID NOs: 1-336 and (SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF,(SEQ ID NO: 342) LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343)PVTOEFWDNLEKETEGLROEMS, (SEQ ID NO: 344) KDLEEVKAKVQ, (SEQ ID NO: 345)KDLEEVKAKVO, (SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN,  (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS,(SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.

In some embodiments, the personalized neoplasia vaccine iscoadministered with an an anti-immunosuppressive or immuno stimulatoryagent. In some embodiments, the anti-immunosuppressive or immunostimulatory agent is selected from the group consisting of anti-CTLA-4antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA,anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40,anti-GITR, anti-ICOS, anti-TIGIT, and inhibitors of IDO.

In certain embodiments, the present invention provides a compositioncomprising a microparticle or nanoparticle associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) one ormore neo-antigenic peptides, wherein each of the one or moreneo-antigenic peptides is specific for a neo-antigenic mutationidentified from a neoplasia biological sample obtained from a subject.In some embodiments, the subject is a human being.

In some embodiments, the size of the microparticle is between 0.5microns to 100 microns. In some embodiments, the average size of thenanoparticle is between 6 to 500 nm.

In some embodiments, the one or more neo-antigenic peptides range fromabout 5 to about 50 amino acids in length. In some embodiments, the oneor more neo-antigenic peptides range from about 15 to about 35 aminoacids in length. In some embodiments, the one or more neo-antigenicpeptides range from about 18 to about 30 amino acids in length. In someembodiments, the one or more neo-antigenic peptides range from about 6to about 15 amino acids in length.

In some embodiments, the nanoparticle is further associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) withan adjuvant. In some embodiments, the adjuvant is selected from thegroup consisting of CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts(for example, aluminum hydroxide, aluminum phosphate), Amplivax, BCG,CP-870,893, CpG7909, CyaA, dSLIM, Cytokines (such as GM-CSF, IL-2,IFN-a, Flt-3L), IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGAmicroparticles, imiquimod, resiquimod, gardiquimod, 3M-052, SRL172,Virosomes and other Virus-like particles, YF-17D, VEGF trap,beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA),3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands(such as C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide,α-mannosylceramide, α-fructosylceramide, β-galatosylceramide,β-mannosylceramide), STING agonists (e.g. cyclic dinucleotides,including Cyclic [G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p],Cyclic [G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes. AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), andbacterial toxins (such as CT, and LT). In some embodiments, the adjuvantis any derivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof.

In some embodiments, the nanoparticle is a sHDL nanoparticle. In someembodiments, the sHDL nanoparticle comprises a mixture of at least onephospholipid and at least one HDL apolipoprotein or apolipoproteinmimetic. In some embodiments, the HDL apolipoprotein is selected fromthe group consisting of apolipoprotein A-I (apo A-I), apolipoproteinA-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs),and apolipoprotein E (apo E). In some embodiments, the phospholipid isselected from the group consisting of dipalmitoylphosphatidylcholine(DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof. In some embodiments,the HDL apolipoprotein mimetic is an ApoA-I mimetic.

In some embodiments, the ApoA-I mimetic is described by any of SEQ IDNOs: 1-336and

(SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF, (SEQ ID NO: 342)LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343) PVTOEFWDNLEKETEGLROEMS,(SEQ ID NO: 344) KDLEEVICAKVQ, (SEQ ID NO: 345) KDLEEVKAKVO,(SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS,(SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.

In some embodiments, the average particle size of the sHDL nanoparticleis between 6-70 nm.

Moreover, the present invention relates to nanoparticles associated with(e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, admixed)biomacromolecule agents configured for treating, preventing orameliorating various types of disorders, and methods of synthesizing thesame. In particular, the present invention is directed to compositionscomprising synthetic high density lipoprotein (sHDL) nanoparticlescarrying biomacromolecule agents (e.g., nucleic acid, peptides,glycolipids, etc.), methods for synthesizing such sHDL nanoparticles, aswell as systems and methods utilizing such sHDL nanoparticles (e.g., indiagnostic and/or therapeutic settings).

As such, in certain embodiments, the present invention provides methodsfor inhibiting a target gene in a cell, comprising introducing into thecell a composition comprising siRNA encapsulated within a sHDLnanoparticle, wherein the siRNA is capable of inhibiting the target geneby RNA interference, wherein the siRNA comprises two RNA strands thatare complementary to each other. In some embodiments, the siRNA ismodified with cholesterol at the 3′ sense strand. In some embodiments,the cell is in vivo, in vitro, or ex vivo. In some embodiments, the cellis within a human being. In some embodiments, an imaging agent isencapsulated within the sHDL nanoparticle.

In certain embodiments, the present invention provides methods forreducing serum LDL-C levels in patient, comprising administering to thepatient a therapeutically effective amount of a pharmaceuticalcomposition comprising a PCSK9 siRNA encapsulated within a nanoparticle,wherein the PCSK9 siRNA is capable of inhibiting the PCSK9 gene by RNAinterference, wherein the PCSK9 siRNA comprises two RNA strands that arecomplementary to each other, wherein inhibiting of the PCSK9 generesults in reduction of serum LDL-C levels in the patient. In someembodiments, the patient is a human patient. In some embodiments, thePCSK9 siRNA is modified with cholesterol at the 3′ sense strand. In someembodiments, an imaging agent is encapsulated within the nanoparticle.In some embodiments, the nanoparticle is selected from the groupconsisting of sHDL nanoparticle, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle.

In certain embodiments, the present invention provides methods fortreating coronary heart disease in a patient through reducing serumLDL-C levels in the patient, comprising administering to the patient atherapeutically effective amount of a pharmaceutical compositioncomprising a PCSK9 siRNA encapsulated within a nanoparticle, wherein thePCSK9 siRNA is capable of inhibiting the PCSK9 gene by RNA interference,wherein the PCSK9 siRNA comprises two RNA strands that are complementaryto each other, wherein inhibiting of the PCSK9 gene results in reductionof serum LDL-C levels. In some embodiments, the patient is a humanpatient. In some embodiments, the PCSK9 siRNA is modified withcholesterol at the 3′ sense strand. In some embodiments, an imagingagent is encapsulated within the nanoparticle. In some embodiments, thenanoparticle is selected from the group consisting of sHDL nanoparticle,fullerenes, endohedral metallofullerenes buckyballs, trimetallic nitridetemplated endohedral metallofullerenes, single-walled and multi-walledcarbon nanotubes, branched and dendritic carbon nanotubes, goldnanorods, silver nanorods, single-walled and multi-walled boron/nitratenanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohornpeapods, liposomes, nanoshells, dendrimers, any nanostructures,microstructures, or their derivatives formed using layer-by-layerprocesses, self-assembly processes, or polyelectrolytes, microparticles,quantum dots, superparamagnetic nanoparticles, nanorods, cellulosenanoparticles, glass and polymer micro- and nano-spheres, biodegradablePLGA micro- and nano-spheres, gold nanoparticles, silver nanoparticles,carbon nanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle. In someembodiments, the sHDL nanoparticle comprises a mixture of at least onephospholipid and at least one HDL apolipoprotein or apolipoproteinmimetic.

In certain embodiments, the present invention provides methods forinducing a natural killer T cell-mediated immune response in a cellcomprising exposing the cell to a composition comprising an αGalCerglycolipid associated with (e.g., complexed, conjugated, encapsulated,absorbed, adsorbed, admixed) within a nanoparticle, wherein suchexposure results in the induction of a natural killer T cell-mediatedimmune response. In some embodiments, the cell is an in vivo cell, an exvivo cell, or an in vitro cell. In some embodiments, the nanoparticle isselected from the group consisting of sHDL nanoparticle, fullerenes,endohedral metallofullerenes buckyballs, trimetallic nitride templatedendohedral metallofullerenes, single-walled and multi-walled carbonnanotubes, branched and dendritic carbon nanotubes, gold nanorods,silver nanorods, single-walled and multi-walled boron/nitrate nanotubes,carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods,liposomes, nanoshells, dendrimers, any nanostructures, microstructures,or their derivatives formed using layer-by-layer processes,self-assembly processes, or polyelectrolytes, microparticles, quantumdots, superparamagnetic nanoparticles, nanorods, cellulosenanoparticles, glass and polymer micro- and nano-spheres, biodegradablePLGA micro- and nano-spheres, gold nanoparticles, silver nanoparticles,carbon nanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle.

In certain embodiments, the present invention provides methods forinducing an immune response to an antigen comprising administering to asubject in need an effective amount of a composition comprising annanoparticle, wherein the antigen is associated with (e.g., complexed,conjugated, encapsulated, absorbed, adsorbed, admixed) the nanoparticle,wherein an adjuvant is associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) with the nanoparticle.

In some embodiments, the antigen is against PCSK9.

In some embodiments, the antigen is against gp100 melanoma.

In some embodiments, the antigen is selected from the group consistingof a peptide based antigen, a protein based antigen, a polysaccharidebased antigen, a saccharide based antigen, a lipid based antigen, aglycolipid based antigen, a nucleic acid based antigen, an inactivatedorganism based antigen, an attenuated organism based antigen, a viralantigen, a bacterial antigen, a parasite antigen, an antigen derivedfrom an allergen, and a tumor antigen.

In some embodiments, the antigen is a tumor antigen selected from thegroup consisting of alpha-actinin-4, Bcr-Abl fusion protein, Casp-8,beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2,ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein,HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP,myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras,Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel,Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17,SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase,TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE,NY-ESO (LAGS), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), human EGFR proteinor its fragments, such as human EGFR residues 306-325(SCVRACGADSYEMEEDGVRK (SEQ ID NO:374)) and residues 897-915(VWSYGVTVWELMTFGSKPY (SEQ ID NO:375)), HTgp-175, M344, MA-50, MG7-Ag,MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 bindingprotein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, WT1(and WT1-derived peptide sequences: WT1 126-134 (RMFP NAPYL (SEQ IDNO:376)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO:377)), and WT1122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO:378)), MUC1 (andMUC1-derived peptides and glycopeptides such as RPAPGS (SEQ ID NO:379),PPAHGVT (SEQ ID NO:380), and PDTRP (SEQ ID NO:381)), LMP2, EGFRvIII,Idiotype, GD2, Ras mutant, p53 mutant, Proteinase3 (PR1), Survivin,hTERT, Sarcoma translocation breakpoints, EphA2, EphA4, LMW-PTP, PAP,ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgenreceptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, FucosylGM1, Mesothelin, sLe(animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH,NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Spermprotein 17, LCK, HMWMAA, AKAP-4, XAGE 1, B7H3, Legumain, Tie 2, Page4,VEGFR2, MAD-CT-1, FAP, PDGFR-alpha, PDGFR-β, MAD-CT-2, Fos-relatedantigen 1, ERBB2, Folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K,fms-related tyro-sine kinase 1 (FLT1, best known as VEGFR1), KDR, PADRE,TA-CIN (recombinant HPV16 L2E7E6), SOX2, aldehyde dehydrogenase, and anyderivative thereof.

In some embodiments, the antigen is any type of viral, bacterial orself-antigen including, but not limited to, FimH against urinary tractinfection; soluble F protein from respiratory syncytial virus (RSV);NEF, GAG, and ENV protein from HIV; Streptococcus pneumoniae proteins;HMGB1 protein; hemagglutinin and neuroamidase protein against influenza;Viral antigens derived from HPV type 16 and 18; gL2, ICP4, gD2ΔTMR,gD2ΔTMR, or ICP4.2 from HSV-2; antigens from S. pneumoniae, such as apneumolysoid, Choline-binding protein A (CbpA), or Pneumococcal surfaceprotein A (PspA), SP1912, SP1912, SP1912L, SP0148 with or without asignal sequence, SP2108 with or without a signal sequence; Antigens fromChlamydia trachomatis, such as a CT209 polypeptide antigen, a CT253polypeptide antigen, a CT425 polypeptide antigen, a CT497 polypeptideantigen, and a CT843 polypeptide antigen; amyloid-beta peptide.

In some embodiments, the adjuvant is a dendritic cell targetingmolecule. In some embodiments, the adjuvant is selected from the groupconsisting of CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts (forexample, aluminum hydroxide, aluminum phosphate), Amplivax, BCG,CP-870,893, CpG7909, CyaA, dSLIM, Cytokines (such as GM-CSF, IL-2,IFN-a, Flt-3L), IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGAmicroparticles, imiquimod, resiquimod, gardiquimod, 3M-052, SRL172,Virosomes and other Virus-like particles, YF-17D, VEGF trap,beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA),3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands(such as C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide,α-mannosylceramide, α-fructosylceramide, β-galatosylceramide,β-mannosylceramide), STING agonists (e.g. cyclic dinucleotides,including Cyclic [G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p],Cyclic [G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), andbacterial toxins (such as CT, and LT). In some embodiments, the adjuvantis any derivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof.

In certain embodiments, the present invention provides methods forinducing an immune response to an antigen comprising administering to asubject in need an effective amount of a composition comprising ananoparticle, wherein the antigen is associated with (e.g., complexed,conjugated, encapsulated, absorbed, adsorbed, admixed) the nanoparticle.In some embodiments, the antigen is against PCSK9. In some embodiments,the nanoparticle is further associated with (e.g., complexed,conjugated, encapsulated, absorbed, adsorbed, admixed) with an adjuvant.In some embodiments, the nanoparticle is co-administered with anadjuvant.

In some embodiments, the nanoparticle is selected from the groupconsisting of sHDL nanoparticle, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle.

In some embodiments, the adjuvant is a dendritic cell targetingmolecule. In some embodiments, the adjuvant is selected from the groupconsisting of CPG, polylC, poly-ICLC, 1018 ISS, aluminum salts (forexample, aluminum hydroxide, aluminum phosphate), Amplivax, BCG,CP-870,893, CpG7909, CyaA, dSLIM, Cytokines (such as GM-CSF, IL-2,IFN-a, Flt-3L), IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGAmicroparticles, imiquimod, resiquimod, gardiquimod, 3M-052, SRL172,Virosomes and other Virus-like particles, YF-17D, VEGF trap,beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA),3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands(such as C20:2, OCH, α-galatosylceramide, α-C-galatosylceramide,α-mannosylceramide, α-fructosylceramide, β-galatosylceramide,β-mannosylceramide), STING agonists (e.g. cyclic dinucleotides,including Cyclic [G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p],Cyclic [G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), andbacterial toxins (such as CT, and LT). In some embodiments, the adjuvantis any derivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof.

In some embodiments, the antigen is conjugated to the outer surface ofthe nanoparticle. In some embodiments, the adjuvant is conjugated to theouter surface of the nanoparticle. In some embodiments, the adjuvant isencapsulated within the nanoparticle.

In some embodiments, the composition is co-administered with achemotherapeutic agent. In some embodiments, the chemotherapeutic agentis one or more of the following: aldesleukin, altretamine, amifostine,asparaginase, bleomycin, capecitabine, carboplatin, carmustine,cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine,dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol,epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil,gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide,interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin,megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane,mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine,prochloroperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride,trastuzumab, vinblastine, vincristine and vinorelbine tartrate.

In certain embodiments, the present invention provides compositionscomprising a nanoparticle, wherein an antigen is associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) thenanoparticle. In some embodiments, the nanoparticle is furtherassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) with an adjuvant. In some embodiments, the antigen isassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the adjuvant. In some embodiments, the antigen is notassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the adjuvant. In some embodiments, the antigen isconjugated with a hydrophobic molecule. In some embodiments, theadjuvant is conjugated with a hydrophobic molecule. In some embodiments,the average size of the nanoparticle is between 6 to 500 nm.

In some embodiments, the hydrophobic molecule is a lipid molecule. Insome embodiments, the lipid molecule is a membrane-forming lipidmolecule. In some embodiments, the lipid molecule molecule is anon-membrane-forming lipid molecule.

Examples of lipid molecules applicable with the embodiments of thepresent invention include, but are not limited to, phospholipids such aslecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Other non-limiting examples of lipid molecules include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.

Other examples of lipid molecules suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

Other examples of lipid molecules suitable for use in the presentinvention include fatty acids and derivatives or analogs thereof. Theyinclude oleic acid, lauric acid, capric acid (n-decanoic acid), myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,caprylic acid, arachidonic acid, glycerol 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkylesters thereof (e.g., methyl, isopropyl and t-butyl), and mono- anddi-glycerides thereof (i.e., oleate, laurate, caprate, myristate,palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri etal., J. Pharm. Pharmacol., 1992, 44, 651-654).

Other examples of lipid molecules suitable for use in the presentinvention include a lipid molecule modified with PEG (PEG-lipid).Examples of PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes. AdditionalPEG-lipids include, without limitation, PEG-C-DOMG, 2 KPEG-DMG, and amixture thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). OtherPEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150(e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipidconjugates of the present invention. The disclosures of these patentsare herein incorporated by reference in their entirety for all purposes.In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CH₂COOH) isparticularly useful for preparing PEG-lipid conjugates including, e.g.,PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

In some embodiments, the antigen is against PCSK9.

In some embodiments, the antigen is against gp100 melanoma.

In some embodiments, the antigen is selected from the group consistingof a peptide based antigen, a protein based antigen, a polysaccharidebased antigen, a saccharide based antigen, a lipid based antigen, aglycolipid based antigen, a nucleic acid based antigen, an inactivatedorganism based antigen, an attenuated organism based antigen, a viralantigen, a bacterial antigen, a parasite antigen, an antigen derivedfrom an allergen, and a tumor antigen.

In some embodiments, the antigen is a tumor antigen selected from thegroup consisting of alpha-actinin-4, Bcr-Abl fusion protein, Casp-8,beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2,ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein,HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP,myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras,Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel,Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17,SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase,TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE,NY-ESO (LAGS), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), human EGFR proteinor its fragments, such as human EGFR residues 306-325(SCVRACGADSYEMEEDGVRK (SEQ ID NO:374)) and residues 897-915(VWSYGVTVWELMTFGSKPY (SEQ ID NO:375)), HTgp-175, M344, MA-50, MG7-Ag,MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 bindingprotein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, WT1(and WT1-derived peptide sequences: WT1 126-134 (RMFP NAPYL (SEQ IDNO:376)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO:377)), and WT1122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO:378)), MUC1 (andMUC1-derived peptides and glycopeptides such as RPAPGS (SEQ ID NO:379),PPAHGVT (SEQ ID NO:380), and PDTRP (SEQ ID NO:381))), LMP2, EGFRvIII,Idiotype, GD2, Ras mutant, p53 mutant, Proteinase3 (PR1), Survivin,hTERT, Sarcoma translocation breakpoints, EphA2, EphA4, LMW-PTP, PAP,ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgenreceptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, FucosylGM1, Mesothelin, sLe(animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH,NY-BR-1, RGSS, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Spermprotein 17, LCK, HMWMAA, AKAP-4, XAGE 1, B7H3, Legumain, Tie 2, Page4,VEGFR2, MAD-CT-1, FAP, PDGFR-alpha, PDGFR-β, MAD-CT-2, Fos-relatedantigen 1, ERBB2, Folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K,fms-related tyro-sine kinase 1 (FLT1, best known as VEGFR1), KDR, PADRE,TA-CIN (recombinant HPV16 L2E7E6), SOX2, and aldehyde dehydrogenase.

In some embodiments, the antigen is any type of viral, bacterial orself-antigen including, but not limited to, FimH against urinary tractinfection; soluble F protein from respiratory syncytial virus (RSV);NEF, GAG, and ENV protein from HIV; Streptococcus pneumoniae proteins;HMGB1 protein; hemagglutinin and neuroamidase protein against influenza;Viral antigens derived from HPV type 16 and 18; gL2, ICP4, gD2ΔTMR,gD2ΔTMR, or ICP4.2 from HSV-2; antigens from S. pneumoniae, such as apneumolysoid, Choline-binding protein A (CbpA), or Pneumococcal surfaceprotein A (PspA), SP1912, SP1912, SP1912L, SP0148 with or without asignal sequence, SP2108 with or without a signal sequence; Antigens fromChlamydia trachomatis, such as a CT209 polypeptide antigen, a CT253polypeptide antigen, a CT425 polypeptide antigen, a CT497 polypeptideantigen, and a CT843 polypeptide antigen; amyloid-beta peptide.

In some embodiments, the adjuvant is a dendritic cell targetingmolecule. In some embodiments, the adjuvant is an immunstimulatory agentthat activates dendritic cells. CPG, polylC, poly-ICLC, 1018 ISS,aluminum salts (for example, aluminum hydroxide, aluminum phosphate),Amplivax, BCG, CP-870,893, CpG7909, CyaA, dSLIM, Cytokines (such asGM-CSF, IL-2, IFN-a, Flt-3L), IC30, IC31, Imiquimod, ImuFact IMP321, ISPatch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipidA, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system,PLGA microparticles, imiquimod, resiquimod, gardiquimod, 3M-052, SRL172,Virosomes and other Virus-like particles, YF-17D, VEGF trap,beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA),3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands(such as C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide,α-mannosylceramide, α-fructosylceramide, β-galatosylceramide,β-mannosylceramide), STING agonists (e.g. cyclic dinucleotides,including Cyclic [G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p],Cyclic [G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), andbacterial toxins (such as CT, and LT). In some embodiments, the adjuvantis any derivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof.

In some embodiments, the antigen is conjugated to the outer surface ofthe nanoparticle. In some embodiments, the adjuvant is conjugated to theouter surface of the nanoparticle. In some embodiments, the adjuvant isencapsulated within the nanoparticle.

In some embodiments, the nanoparticle is selected from the groupconsisting of sHDL nanoparticle, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle.

In certain embodiments, the present invention provides comprising siRNAencapsulated within a nanoparticle, wherein the siRNA is capable ofinhibiting a target gene by RNA interference, wherein the siRNAcomprises two RNA strands that are complementary to each other. In someembodiments, the siRNA is modified with cholesterol at the 3′ sensestrand. In some embodiments, an imaging agent is encapsulated within thenanoparticle.

In some embodiments, the nanoparticle is selected from the groupconsisting of sHDL nanoparticle, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle.

In certain embodiments, the present invention provides comprising aPCSK9 siRNA encapsulated within a nanoparticle, wherein the PCSK9 siRNAis capable of inhibiting the PCSK9 gene by RNA interference, wherein thePCSK9 siRNA comprises two RNA strands that are complementary to eachother. In some embodiments, the PCSK9 siRNA is modified with cholesterolat the 3′ sense strand. In some embodiments, an imaging agent isencapsulated within the nanoparticle.

In some embodiments, the average size of the nanoparticle is between 6to 500 nm.

In some embodiments, the nanoparticle is selected from the groupconsisting of sHDL nanoparticle, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, a modified micelle. In someembodiments, the nanoparticle is a sHDL nanoparticle.

In certain embodiments, the present invention provides comprising anαGalCer glycolipid associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) a nanoparticle.

Such methods and compositions are not limited to particular size, typeor kind of nanoparticles. In some embodiments, the nanoparticle isselected from the group consisting of sHDL nanoparticle, fullerenes,endohedral metallofullerenes buckyballs, trimetallic nitride templatedendohedral metallofullerenes, single-walled and multi-walled carbonnanotubes, branched and dendritic carbon nanotubes, gold nanorods,silver nanorods, single-walled and multi-walled boron/nitrate nanotubes,carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods,liposomes, nanoshells, dendrimers, any nanostructures, microstructures,or their derivatives formed using layer-by-layer processes,self-assembly processes, or polyelectrolytes, microparticles, quantumdots, superparamagnetic nanoparticles, nanorods, cellulosenanoparticles, glass and polymer micro- and nano-spheres, biodegradablePLGA micro- and nano-spheres, gold nanoparticles, silver nanoparticles,carbon nanoparticles, iron nanoparticles, a modified micelle.

In some embodiments, the nanoparticle is a sHDL nanoparticle. In someembodiments, the sHDL nanoparticle comprises a mixture of at least onephospholipid and at least one HDL apolipoprotein or apolipoproteinmimetic.

In some embodiments, the HDL apolipoprotein is selected from the groupconsisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apoA-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), andapolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein isselected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I,preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-IV,ApoA-V, preproApoE, proApoE, ApoE, preproApoA-IMilano, proApoA-IMilanoApoA-IMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis andpeptide mimetics of these proteins mixtures thereof.

In some embodiments, the phospholipid is selected from the groupconsisting of dipalmitoylphosphatidylcholine (DPPC),dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof.

In some embodiments, the HDL apolipoprotein mimetic is an ApoA-Imimetic.

In some embodiments, the ApoA-I mimetic is described by any of SEQ IDNOs: 1-336 and

(SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF, (SEQ ID NO: 342)LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343) PVTOEFWDNLEKETEGLROEMS,(SEQ ID NO: 344) KDLEEVKAKVQ, (SEQ ID NO: 345) KDLEEVKAKVO,(SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS,(SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.

In some embodiments, the average particle size of the sHDL nanoparticleis between 6-70 nm.

In certain embodiments, the present invention provides methods forinducing an immune response to one or more antigens comprisingadministering to a subject in need an effective amount of a compositioncomprising a nanoparticle, wherein the one or more antigens isassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the nanoparticle, wherein an adjuvant is associatedwith (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed,admixed) the nanoparticle. In some embodiments, the antigen isassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the adjuvant. In some embodiments, the antigen is notassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the adjuvant. In some embodiments, the antigen isconjugated with a hydrophobic molecule. In some embodiments, theadjuvant is conjugated with a hydrophobic molecule. In some embodiments,the average size of the nanoparticle is between 6 to 500 nm.

In certain embodiments, the present invention provides compositionscomprising a nanoparticle, wherein one or more antigens is associatedwith (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed,admixed) the nanoparticle, wherein an adjuvant is associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) thenanoparticle. In some embodiments, the antigen is associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) theadjuvant. In some embodiments, the antigen is not associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) theadjuvant. In some embodiments, the antigen is conjugated with ahydrophobic molecule. In some embodiments, the adjuvant is conjugatedwith a hydrophobic molecule. In some embodiments, the average size ofthe nanoparticle is between 6 to 500 nm.

In some embodiments, the hydrophobic molecule is a lipid molecule. Insome embodiments, the lipid molecule is a membrane-forming lipidmolecule. In some embodiments, the lipid molecule molecule is anon-membrane-forming lipid molecule.

Examples of lipid molecules applicable with the embodiments of thepresent invention include, but are not limited to, phospholipids such aslecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Other non-limiting examples of lipid molecules include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.

Other examples of lipid molecules suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

Other examples of lipid molecules suitable for use in the presentinvention include fatty acids and derivatives or analogs thereof. Theyinclude oleic acid, lauric acid, capric acid (n-decanoic acid), myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,caprylic acid, arachidonic acid, glycerol 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkylesters thereof (e.g., methyl, isopropyl and t-butyl), and mono- anddi-glycerides thereof (i.e., oleate, laurate, caprate, myristate,palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 921 Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri etal., J. Pharm. Pharmacol., 1992, 44, 651-654).

Other examples of lipid molecules suitable for use in the presentinvention include a lipid molecule modified with PEG (PEG-lipid).Examples of PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes. AdditionalPEG-lipids include, without limitation, PEG-C-DOMG, 2 KPEG-DMG, and amixture thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). OtherPEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150(e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipidconjugates of the present invention. The disclosures of these patentsare herein incorporated by reference in their entirety for all purposes.In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CH₂COOH) isparticularly useful for preparing PEG-lipid conjugates including, e.g.,PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.

Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

In some embodiments, the one or more antigens is against PCSK9, M30,M27, Adpgk, and ASMTNMELM (SEQ ID NO:383). In some embodiments, the oneor more antigens are conjugated to the outer surface of thenanoparticle.

In some embodiments, the adjuvant is selected from the group consistingof CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts (for example,aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870,893,CpG7909, CyaA, dSLIM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L),IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGA microparticles,imiquimod, resiquimod, gardiquimod, 3M-052, SRL172, Virosomes and otherVirus-like particles, YF-17D, VEGF trap, beta-glucan, Pam3Cys, Aquila'sQS21 stimulon, vadimezan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyllipid adjuvant (GLA), GLA-SE, CD1d ligands (such as C20:2, OCH,α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide,α-fructosylceramide, β-galatosylceramide, β-mannosylceramide), STINGagonists (e.g. cyclic dinucleotides, including Cyclic[G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p], Cyclic[G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule. TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), andbacterial toxins (such as CT, and LT). In some embodiments, the adjuvantis any derivative of an adjuvant (e.g., cholesterol-modified CpG) or anycombinations thereof. In some embodiments, the adjuvant is conjugated tothe outer surface of the nanoparticle. In some embodiments, the adjuvantis encapsulated within the nanoparticle.

In some embodiments, the nanoparticle is selected from the groupconsisting of sHDL nanoparticle, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, a modified micelle.

In some embodiments, the nanoparticle is a sHDL nanoparticle. In someembodiments, the nanoparticle is sHDL, wherein the sHDL nanoparticlecomprises a mixture of at least one phospholipid and at least one HDLapolipoprotein or apolipoprotein mimetic.

In some embodiments, the HDL apolipoprotein is selected from the groupconsisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apoA-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), andapolipoprotein E (apo E), wherein the phospholipid is selected from thegroup consisting of dipalmitoylphosphatidylcholine (DPPC),dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof.

In some embodiments, the HDL apolipoprotein mimetic is an ApoA-Imimetic, wherein the thiol-reactive phospholipid isdioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP).

In some embodiments, the ApoA-I mimetic is described by any of SEQ IDNos: 1-336and

(SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF, (SEQ ID NO: 342)LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343) PVTOEFWDNLEKETEGLROEMS,(SEQ ID NO: 344) KDLEEVKAKVQ, (SEQ ID NO: 345) KDLEEVKAKVO,(SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS,(SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.

Full-length apolipoprotein (typically over 20KDa) could be producedeither by purification from out-dated plasma or by recombinantmethodology. In contrast to full-length apolipoproteins, peptides asshort as 18-25 amino acids are capable to form α-helix and could beproduced by synthetic methodologies. Due to complexity of proteins, thecost associated with protein-based therapeutics (e.g. processdevelopment, manufacturing, and analysis) is typically 10 to 100 foldhigher than peptide-based therapeutics. Additional experiments conductedduring the course of developing embodiments for the present inventioncompared the use of apolipoprotein peptide with full lengthapolipoprotein. Such experiments demonstrate the advantages of utilizingapolipoprotein peptide in relation to manufacturing, ease of sHDLformation, and reduced manufacturing cost relative to utilization offull length apolipoprotein (see, Examples VIII and IX).

To produce recombinant protein, an expression system and a purificationmethod need to be developed in order to produce and purify protein fromhost cell-related impurities. Selection of expression system,development of fermentation, and purification methodology acceptable forscale-up under current Good Manufacturing Practices (cGMP) often take1-2 years and significant financial resources.

Typical cell-culture and purification process for biologics consists ofseveral steps (see, FIGS. 30 and 31). For example, certain methodsinvolve the following steps: thawing of vial of working cell bank,preparation of inoculum (shake flask, small bioreactors), transfer toproduction bioreactor, harvest/filtration or cell destruction,purification with orthogonal chromatography steps, including:ion-exchange chromatography, hydrophobic interaction chromatography,reverse phase chromatography, multiple concentration and buffer exchangesteps, viral filtration, endotoxin/DNA removal filtration, final sterilefiltration and freeze/storage of purified protein. All purificationsteps are required in order to remove impurities.

To produce and purify recombinant apolipoproteins, a large number ofsteps are required to eliminate process- and product-related impurities.The process impurities include: host cell components, such as host cellproteins (HCP), DNA, membrane components, endotoxin, viral adventitiousagents, mycoplasma, microbial adventitious agents, adenoviruses, andplasmid DNA. Their levels have to be measured prior to human use. Otherimpurities include anti-foam agents, antibiotics, surfactants,leachables/extractables, organic solvents and other molecules added tocontrol upstream or downstream processes.

The product-related impurities include: truncated apolipoprotein,apolipoprotein containing leader sequences like (pre and proapolipoprotein A-I), aggregated, oxidized, de-amidated and hydrolyzedprotein.

All impurities have to be controlled at levels specified by FDA, WHO,and ICH guidelines and specification set for apolipoprotein product. Toarchive these specifications, multiple purification steps are required,leading to decreased process yields, longer manufacturing time andhigher manufacturing costs.

To obtain and purify apoliproteins from plasma, the following processneeds to be established. Apoliproproteins can be separated and purifiedfrom human plasma. However, the supply of human plasma is mainlydependent on voluntary blood donation, and the availability of plasmamay limit the manufacturing of apoliproproteins. In addition, thecomplexity of plasma itself also presents great challenges for theseparation and purification of apoliproproteins.

Cold ethanol fractionation of human plasma has also been developed forthe production of apoliproproteins. Precipitates of cold ethanolfractionation are used as starting materials, followed by solubilizationin guanidine hydrochloride and purification by gel filtration andanion-exchange chromatography.

However, for these methods, purification of apoliproproteins requiresthe use of solvent that has a high alkaline environment, which can causepartial degradation of apoliproproteins, triggering immunogenicresponses. U.S. patent application Ser. No. 12/673,723 reported animproved method for apoliproprotein purification from plasma to reduceapoliproprotein degradation and improve the purity (FIG. 32). In thismethod, the fraction IV₁ paste from a cold ethanol fractionationprocess-treated human plasma was used as starting materials forpurification of apoliproprotein. The fraction IV₁ paste was suspended ina suspension buffer (100 mM Tris, pH 9.6), followed by pH/alcoholadjustment with ethanol and sodium acetate/acetic acid solution toprecipitate apoliproprotein from other components. Separatedapoliproprotein was further purified by passing through a cellulosefilter coated with filter aid (Celite™ 574). The purity ofapoliproprotein was reported to be up to 89%, which corresponds topharmaceutical grade purity. Other methods such as ion exchangechromatography and hydrophobic interaction chromatography have also beenused for the purification of apoliproproteins.

The manufacturing process for chemical synthesis ofapolipoprotein-mimetic peptides summarized in FIG. 33. Compared withendogenous apolipoproteins, apolipoprotein-mimetic peptides have severalmajor advantages.

First, the starting materials for apolipoprotein-mimetic peptidesynthesis are amino acids, and they assembled into peptides viasolid-phase peptide synthesis on resin, followed by deprotection andcleavage from the resin and column purification to obtain the peptide(FIG. 34). Since amino acids are cheap and easy to obtain, thelarge-scale chemical synthesis of peptide can be easily achieved.

Second, compared with recombinant production of apolipoproteins andpurification from human plasma, the chemical peptide synthesis issimpler and results in lower levels of product and process impurities.Therefore, it is easier to obtain highly pure form of peptide (>99%purity)—the level that is very challenging to achieve forapolipoproteins. The simplicity of peptides also makes it easier forquality control, compared with apolipoproteins, which may requirecomplex assays to monitor the quality. More importantly, there is norisk of contamination of chemically synthesized peptides by pathogens orother unknown chemicals, which may be present in human plasma. Third,the sequences of apolipoprotein-mimetic peptides can be optimized basedon the binding affinity to lipids and homogeneity of formed HDL.

In certain embodiments, the present invention provides compositionscomprising a nanoparticle (as described herein), wherein any kind ofbiomacromolecule agent (e.g., nucleic acid, peptides, glycolipids, etc.)is associated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the nanoparticle.

In some embodiments, the biomacromolecule agent is a peptide.

For example, in some embodiments, the peptide is an antigen. In someembodiments wherein the peptide is an antigen, the composition furthercomprises an adjuvant (as described herein).

As noted, the peptide is not limited to a particular type of peptide.

In some embodiments, the peptide is Adrenocorticotropic Hormone (ACTH),a growth hormone peptide, a Melanocyte Stimulating Hormone (MSH),Oxytocin, Vasopressin, Corticotropin Releasing Factor (CRF), aCRF-related peptide, a Gonadotropin Releasing Hormone Associated Peptide(GAP), Growth Hormone Releasing Factor (GRF), Lutenizing Hormone ReleaseHormone (LH-RH), an orexin, a Prolactin Releasing Peptide (PRP), asomatostatin, Thyrotropin Releasing Hormone (THR), a THR analog,Calcitonin (CT), a CT-precursor peptide, a Calcitonin Gene RelatedPeptide (CGRP), a Parathyroid Hormone (PTH), a Parathyroid HormoneRelated Protein (PTHrP), Amylin, Glucagon, Insulin, an Insulin-likepeptide, NeuroPeptide Y (NPY), a Pancreatic Polypeptide (PP), Peptide YY(PYY), Cholecystokinin (CCK), a CCK-related peptide, Gastrin ReleasingPeptide (GRP), Gastrin, a Gastrin-related peptide, a Gastrin inhibitorypeptide, Motilin, Secretin, Vasoactive Intestinal Peptide (VIP), aVIP-related peptide, an Atrial-Natriuretic Peptide (ANP), a BrainNatriuretic Peptide (BNP), a C-Type Natriuretic Peptide(CNP), atachykinin, an angiotensin, a renin substrate, a renin inhibitor, anendothelin, an endothelin-related peptide, an opioid peptide, a thymicpeptide, an adrenomedullin peptide, an allostatin peptide, an amyloidbeta-protein fragment, an antimicrobial peptide, an antioxidant peptide,an apoptosis related peptide, a Bag Cell Peptide (BCPs), Bombesin, abone Gla protein peptide, a Cocaine and Amphetamine Related Transcript(CART) peptide, a cell adhesion peptide, a chemotactic peptide, acomplement inhibitor, a cortistatin peptide, a fibronectin fragment, afibrin related peptide, FMRF, a FMRF amide-related peptide (FaRP),Galanin, a Galanin-related peptide, a growth factor, a growthfactor-related peptide, a G-Therapeutic Peptide-Binding Proteinfragment, Gualylin, Uroguanylin, an Inhibin peptide, Interleukin (IL),an Interleukin Receptor protein, a laminin fragment, a leptin fragmentpeptide, a leucokinin, Pituitary Adenylate Cyclase ActivatingPolypeptide (PAPCAP), Pancreastatin, a polypeptide repetitive chain, asignal transducing reagent, a thrombin inhibitor, a toxin, a trypsininhibitor, a virus-related peptide, an adjuvant peptide analog, AlphaMating Factor, Antiarrhythmic Peptide, Anorexigenic Peptide, Alpha-1Antitrypsin, Bovine Pineal Antireproductive Peptide, Bursin, C3 PeptideP16, Cadherin Peptide, Chromogranin A Fragment, ContraceptiveTetrapeptide, Conantokin G, Conantokin T, Crustacean CardioactivePeptide, C-Telopeptide, Cytochrome b588 Peptide, Decorsin, DeliciousPeptide, Delta-Sleep-Inducing Peptide, Diazempam-Binding InhibitorFragment, Nitric Oxide Synthase Blocking Peptide, OVA Peptide, PlateletCalpain Inhibitor (P1), Plasminogen Activator Inhibitor 1, Rigin,Schizophrenia Related Peptide, Sodium Potassium Atherapeutic PeptidaseInhibitor-1, Speract, Sperm Activating Peptide, Systemin, a Thrombinreceptor agonist, Tuftsin, Adipokinetic Hormone, Uremic Pentapeptide,Antifreeze Polypeptide, Tumor Necrosis Factor (TNF), Leech [DesAsp10]Decorsin, L-Omithyltaurine Hydrochloride, P-AminophenylacetylTuftsin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro,Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly,D-Ala-Leu, D-D-D-D-D, D-D-D-D-D-D, N-P-N-A-N-P-N-A, V-A-I-T-V-L-V-K,V-G-V-R-V-R, V-I-H-S, V-P-D-P-R, Val-Thr-Cys-Gly, R-S-R, Sea UrchinSperm Activating Peptide, a SHU-9119 antagonist, a MC3-R antagonist, aMC4-R antagonist, Glaspimod, HP-228, Alpha 2-Plasmin Inhibitor, APCTumor Suppressor, Early Pregnancy Factor, Gamma Interferon, GlandularKallikrei N-1, Placental Ribonuclease Inhibitor, Sarcolecin BindingProtein, Surfactant Protein D, Wilms' Tumor Suppressor, GABAB 1bReceptor Peptide, Prion Related Peptide (iPRP13), Choline BindingProtein Fragment, Telomerase Inhibitor, Cardiostatin Peptide, EndostatinDerived Peptide, Prion Inhibiting Peptide, N-Methyl D-Aspartate ReceptorAntagonist, and C-PeptideAnalog.

In some embodiments, the peptide is selected from177Lu-DOTA0-Tyr3-Octreotate, Abarelix acetate, ADH-1, Afamelanotidec,melanotan-1, CUV1647, Albiglutide, Aprotinin, Argipressin, Atosibanacetate, Bacitracin, Bentiromide, a BH3 domain, Bivalirudin, Bivalirudintrifluoroacetate hydrate, Blisibimod, Bortezomib, Buserelin, Buserelinacetate, Calcitonin, Carbetocin, Carbetocin acetate, Cecropin A and B,Ceruletide, Ceruletide diethylamine, Cetrorelix, Cetrorelix acetate,Ciclosporine, Cilengitidec, EMD121974, Corticorelin acetate injection,hCRF, Corticorelin ovine triflutate, corticorelin trifluoroacetate,Corticotropin, Cosyntropin, ACTH 1-24, tetracosactide hexaacetate,Dalbavancin, Daptomycin, Degarelix acetate, Depreotide trifluoroacetate(plus sodium pertechnetate), Desmopressin acetate, Desmopressin DDAVP,Dulaglutide, Ecallantide, Edotreotide (plus yttrium-90), Elcatoninacetate, Enalapril maleate (or 2-butanedioate), Enfuvirtide,Eptifibatide, Exenatide, Ganirelix acetate, Glatiramer acetate,Glutathion, Gonadorelin, Gonadorelin acetate, GnRH, LHRH, Goserelin,Goserelin acetate, Gramicidin, Histrelin acetate, Human calcitonin,Icatibant, Icatibant acetate, IM862, oglufanide disodium, KLAKLAK,Lanreotide acetate, Lepirudin, Leuprolide, Leuprolide acetate,leuprorelin, Liraglutide, Lisinopril, Lixisenatide, Lypressin,Magainin2, MALP-2Sc, macrophage-activating lipopeptide-2 synthetic,Nafarelin acetate, Nesiritide, NGR-hTNF, Octreotide acetate,Oritavancin, Oxytocin, Pasireotide, Peginesatide, Pentagastrin,Pentetreotide (plus indium-111), Phenypressin, Pleurocidin, Pramlintide,Protirelin, thyroliberin, TRH, TRF, Salmon calcitonin, Saralasinacetate, Secretin (human), Secretin (porcine), Semaglutide, Seractideacetate, ACTH, corticotropin, Sermorelin acetate, GRF 1-29, Sinapultide,KL4 in lucinactant, Sincalide, Somatorelin acetate, GHRH, GHRF, GRF,Somatostatin acetate, Spaglumat magnesium (or sodium) salt, Substance P,Taltirelin hydrate, Teduglutide, Teicoplanin, Telavancin, Teriparatide,Terlipressin acetate, Tetracosactide, Thymalfasin, thymosin a-1,Thymopentin, Trebananib, Triptorelin, Triptorelin pamoate,Tyroserleutide, Ularitide, Vancomycin, Vapreotide acetate, Vasoactiveintestinal peptide acetate, Vx-001c, TERT572Y, Ziconotide acetate, α5-α6Bax peptide, and β-defensin.

In some embodiments, the peptide is any peptide which would assist inachieving a desired purpose with the composition. For example, in someembodiments, the peptide is any peptide that will facilitate treatmentof any type of disease and/or disorder (e.g., peripheral ischemia,cancer, inflammatory disorders, genetic disorders, etc.).

In some embodiments, the biomacromolecule agent is a nucleic acid. Suchembodiments encompass any type of nucleic acid molecule including, butnot limited to, RNA, siRNA, microRNA, interference RNA, mRNA, repliconmRNA, RNA-analogues, and DNA.

In certain embodiments, the present invention provides methods fortreating conditions, disorders and/or diseases with such compositionscomprising a nanoparticle (as described herein), wherein any kind ofbiomacromolecule is associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) the nanoparticle.

The present invention is not limited to specific types of conditions,disorders and/or diseases.

In some embodiments, the condition, disorder and/or disease isperipheral ischemia, cancer, an inflammatory disorder, a geneticdisorder, etc.

In some embodiments, the condition, disorder and/or disease is selectedfrom erythropoietic porphyries, T2 diabetes, antifibrinolytic, centraldiabetes insipidus, delaying the birth in case of threat of prematurebirth, antibiotic, cystic fibrosis, angina, anticoagulant in patientswith unstable angina undergoing PTCA or PCI, systemic lupuserythematosus, hypercalcemia, osteoporosis, pagets disease, carbetocinworks as an oxytocic, antihemorrhagic and uterotonic drug in theperipheral nervous system, prevention of uterine atony, induction, andcontrol postpartum bleeding or hemorrhage, stimulant of the gastricsecretion, for treat hormone-sensitive cancers of the prostate andbreast, inhibition of premature LH surges in women undergoing controlledovarian stimulation, immunosuppression in organ transplantation toprevent rejection, peritumoral brain edema, diagnosis of ACTHdependentCushing's syndrome, allergies, ankylosing spondylitis, psoriasis,chorioditis, erythema, keratitis, sclerosis, dermatomyositis, rheumatoidarthritis, Stevens-Johnson Syndrome, ulcerative colitis, diagnosis ofadrenocortical insufficiency, antibiotic, systemic infections caused bygram positive organisms, nocturnal enuresis, nocturia, and stoppage ofbleeding or hemorrhage in haemophilia A patients, acute hereditaryangioderma, postmenopausal osteoporosis, anti-parathyroid, Paget'sdisease, hypercalcaemia, hypertension, AIDS/HIV-1 infection, acutecoronary syndrome, unstable angina undergoing PCI, Alzheimer's andParkinson's disease, inhibition of premature LH surges in womenundergoing controlled ovarian hyperstimulation, Relapsing-RemittingMultiple Sclerosis, hepatic insufficiency, wound healing, inflammationof respiratory tract, asthenia, release of follicle-stimulating hormone(FSH) and luteinizing hormone (LH) from the anterior pituitary,stimulate the secretion of gonadotropin during disturbances fertility,and diagnosis of the functional capacity and response of thegonadotropes of the anterior pituitary, for skin lesions, surface woundsand eye infections, postmenopausal osteoporosis, Paget's disease,hypercalcaemia, hereditary angioedema, immune system related diseases,acromegaly, anticoagulant, fibroids and endometriosis, central diabetesinsipidus, Cushing's syndrome, diabetic foot ulcers, treatment ofcentral precocious puberty, uterine fibriods and endometriosis,vasodilatory, natriuretic, diuretic and neurohormonal effects,acromegaly, carcinoid syndrome, acute bacterial skin and skin structureinfections, initiation or improvement of uterine contractions, andcontrol postpartum bleeding or hemorrhage, hematide Chronic kidneydisease associated anemia, stomatitis, pharyngitis, diagnosticassessment of thyroid function, postmenopausal osteoporosis,hypercalcaemia, diagnosis of pancreatic exocrine dysfunction, andgastrinoma, Zollinger-Ellison syndrome, prevention of RDS in prematureinfants, and meconium aspiration syndrome, acute variceal bleeding,allergic rhinitis and conjunctivitis, spinocerebellardegeneration/ataxia, Short Bowel Syndrome, antibiotic, bactericidal,teriparatide is the only anabolic (i.e., bone growing) agent indicatedfor use in postmenopausal women with osteoporosis, Cortrosyn Analogue ofadrenocorticotrophic hormone (ACTH) used for diagnostic purposes,treatment of adrenal insufficiency, epilepsia, Chronic hepatitis B,chronic hepatitis C, primary and secondary immune deficiencies, acutedecompensated heart failure, colitis, esophageal variceal bleeding inpatients with cirrhotic liver disease and AIDS-related diarrhea,sarcoidosis and acute lung injury, and severe chronic pain.

In some embodiments, the peptide and condition, disorder and/or diseaseto be treated is shown in each row of Table 1. Table 1 provides a listof therapeutic peptides and conditions, disorders, and/or diseasestargeted by such therapeutic peptides (e.g., each row presents atherapeutic peptide and the condition, disorder and/or disease targetedby the respective therapeutic peptide).

TABLE 1 Therapeutic Peptide Target Condition, Disorder and/or Disease177Lu-DOTA0-Tyr3- Midgut carcinoid tumors Octreotate Abarelix acetateAdvanced prostate cancer ADH-1 Malignant melanoma (FDA orphan drugstatus Phase II) Afamelanotidec, or Erythropoietic porphyries (EMEA andFDA melanotan-1, or orphan drug status Phase III) CUV1647 Albiglutide T2diabetes Aprotinin Antifibrinolytic Argipressin Central diabetesinsipidus, and BOV Atosiban acetate Delaying the birth in case of threatof premature birth Bacitracin Antibiotic Bentiromide Approved by FDA forscreening test of exocrine pancreatic insufficiency in patients withcystic fibrosis BH3 domain Cancer: apoptosis Bivalirudin Direct thrombininhibitor Bivalirudin Angina Bivalirudin Anticoagulant in patients withunstable angina trifluoroacetate undergoing PTCA or PCI hydrateBlisibimod Systemic lupus erythematosus Bortezomib Multiple myeloma, andrefractory, mantle cell lymphoma Buserelin Treatment of prostate andbreast cancer Buserelin acetate Advanced prostate cancer CalcitoninHypercalcemia, osteoporosis, pagets disease Carbetocin Carbetocin worksas an oxytocic, antihemorrhagic and uterotonic drug in the peripheralnervous system Carbetocin acetate Prevention of uterine atony,induction, and control postpartum bleeding or haemorrhage Cecropin A andB Leukemia; Bladder cancer Ceruletide Potent cholecystokinetic agentwith a direct spasmogenic effect on the gallbladder muscle and bileducts in humans and animals Ceruletide Diagnosis of the functional stateof the diethylamine gallbladder and pancreas, and stimulant of thegastric secretion Cetrorelix GnRH antagonist, used to treat hormone-sensitive cancers of the prostate and breast Cetrorelix acetateInhibition of premature LH surges in women undergoing controlled ovarianstimulation Ciclosporine An immunosuppressant drug widely used in organtransplantation to prevent rejection Cilengitidec, or GBM (EMEA and FDAorphan drug status- EMD121974 Phase III) Corticorelin acetatePeritumoral brain edema (FDA orphan drug injectionc, or hCRFstatus-Phase III) Corticorelin ovine Diagnosis of ACTHdependentCushing's triflutate, or syndrome corticorelin trifluoroacetateCorticotropin Allergies, ankylosing spondylitis, psoriasis, chorioditis,erythema, keratitis, sclerosis, dermatomyositis, rheumatoid arthritis,Stevens- Johnson Syndrome, Systemic Lupus Erythematosus, UlcerativeColitis Cosyntropin, or Diagnosis of adrenocortical insufficiency ACTH1-24, or tetracosactide hexaacetate Dalbavancin antibiotic DaptomycinSystemic infections caused by gram positive organisms Degarelix acetateTreatment of prostate cancer Depreotide Diagnosis (scintigraphicimaging) of lung trifluoroacetate tumours (plus sodium pertechnetate)Desmopressin acetate insipidus, nocturnal enuresis, nocturia, andstoppage of bleeding or haemorrhage in haemophilia A patientsDesmopressin To treat nocturnal enuresis (bedwetting) DDAVP DulaglutideType 2 diabetes mellitus Ecallantide Acute hereditary angiodermaEdotreotide (plus Gastro-entero-pancreatic neuroendocrine yttrium-90)tumours (FDA orphan drug status-Phase II) Elcatonin acetatePostmenopausal osteoporosis, anti-parathyroid, Paget's disease,hypercalcaemia Enalapril maleate Hypertension (or 2-butanedioate)Enfuvirtide AIDS/HIV-1 infection Eptifibatide Acute coronary syndrome,unstable angina undergoing PCI Exenatide Type 2 diabetes. PreclinicalStudies also revealed its neuroprotective role in Alzheimer's andParkinson's disease Ganirelix acetate Inhibition of premature LH surgesin women undergoing controlled ovarian hyperstimulation Glatirameracetate Reduction of the frequency of relapses in patients withRelapsing-Remitting Multiple Sclerosis Glutathion Hepatic insufficiency,wound healing, inflammation of respiratory tract, asthenia GonadorelinRelease of follicle-stimulating hormone (FSH) and luteinizing hormone(LH) from the anterior pituitary Gonadorelin acetate, or Stimulate thesecretion of gonadotropin during GnRH, or LHRH disturbances fertility,and diagnosis of the functional capacity and response of thegonadotropes of the anterior pituitary Goserelin Breast cancer andprostate cancer Goserelin acetate Advanced prostate cancer, breastcancer Gramicidin Used to treat skin lesions, surface wounds and eyeinfections Histrelin acetate Treatment of prostate and breast cancerHuman calcitonin Postmenopausal osteoporosis, Paget's disease,hypercalcaemia Icatibant Hereditary angioedema Icatibant acetateHereditary angioedema IM862, or oglufanide Immune system relateddiseases (FDA orphan disodium drug status for ovarian cancer-Phase II)KLAKLAK glioblastoma Lanreotide acetate Acromegaly LepirudinAnticoagulant Leuprolide Treatment of prostate and breast cancer, and totreat fibroids and endometriosis, Alzheimer disease Leuprolide acetate,or Advanced prostate cancer, breast cancer, central leuprorelinprecocious puberty Liraglutide Type 2 diabetes Lisinopril Hypertension,congestive heart failure Lixisenatide Treatment of Diabetes LypressinCentral diabetes insipidus, Cushing's syndrome Magainin2 Bladder cancer;Diabetic foot ulcers MALP-2Sc, or Pancreatic cancer (EMEA orphan drugstatus- macrophage-activating Phase II) lipopeptide-2 syntheticNafarelin acetate Treatment of central precocious puberty, uterinefibriods and endometriosis Nesiritide Vasodilatory, natriuretic,diuretic and neurohormonal effects NGR-hTNF Mesothelioma Octreotideacetate Acromegaly, carcinoid syndrome Oritavancin Acute bacterial skinand skin structure infections Oxytocin Initiation or improvement ofuterine contractions, and control postpartum bleeding or haemorrhagePasireotide Cushing's disease and Acromegaly Peginesatide HematideChronic kidney disease associated anemia Pentagastrin Diagnosis of thegastric secretion Pentetreotide (plus Diagnosis (scintigraphic imaging)of primary and indium-111) metastatic neuroendocrine tumoursPhenypressin Stomatitis, pharyngitis Pleurocidin Breast cancerPramlintide Diabetes Protirelin, or Diagnostic assessment of thyroidfunction thyroliberin, or TRH, or TRF Salmon calcitonin Postmenopausalosteoporosis, Paget's disease, hypercalcaemia Saralasin acetateHypertension Secretin (human) Diagnosis of pancreatic exocrinedysfunction, and gastrinoma, Zollinger-Ellison syndrome Secretin(porcine) Diagnosis of pancreatic exocrine dysfunction, and gastrinoma,Zollinger-Ellison syndrome Semaglutide T2 diabetes Seractide acetate, orDiagnosis of adrenocortical insufficiency ACTH, or corticotropinSermorelin acetate or Growth hormone deficiency, diagnosis evaluationGRF 1-29 of pituitary function Sinapultide, or Prevention of RDS inpremature infants, and KL4 in lucinactant meconium aspiration syndromeSincalide Diagnosis of the functional state of the gallbladder andpancreas, and stimulant of the gastric secretion Somatorelin Diagnosisof somatotropic function of the anterior acetate, or GHRH, pituitarygland in cases of suspected growth or GHRF, or GRF hormone deficiency(hypophysic and hypothalamic disorders) Somatostatin acetate Acutevariceal bleeding Spaglumat magnesium Allergic rhinitis andconjunctivitis (or sodium) salt Taltirelin hydrate Spinocerebellardegeneration/ataxia Teduglutide Short Bowel Syndrome (EMEA and FDAorphan drug status-Phase III) Teicoplanin Antibiotic Telavancinbactericidal Teriparatide Teriparatide is the only anabolic (i.e., bonegrowing) agent indicated for use in postmenopausal women withosteoporosis Terlipressin acetate BOV Tetracosactide Cortrosyn Analogueof adrenocorticotrophic hormone (ACTH) used for diagnostic purposes,treatment of adrenal insufficiency, different types of drug registantepilepsia Thymalfasin, or Chronic hepatitis B, chronic hepatitis Cthymosin a-1 Thymopentin Primary and secondary immune deficiencies,autoimmunity, infections, cancer Trebananib Ovarian, peritoneal orfallopian tube cancers Triptorelin Trelstar Treatment of prostate andbreast cancer Triptorelin pamoate Advanced prostate cancer, centralprecocious puberty, endometriosis, uterine fibroids, ovarian stimulationin in vitro fecundation Tyroserleutide Hepatocellular carcinomaUlaritide Acute decompensated heart failure Vancomycin Treat colitis(inflammation of the intestine caused by certain bacteria) that mayoccur after antibiotic treatment Vapreotide acetate Treatment ofesophageal variceal bleeding in patients with cirrhotic liver diseaseand AIDS- related diarrhea Vasoactive intestinal Sarcoidosis and acutelung injury (EMEA and peptide acetate FDA orphan drug status Phase II)Vx-001c or TERT572Y NSCLC (EMEA and FDA orphan drug status- Phase II)Ziconotide acetate Severe chronic pain α5-α6 Bax peptide Cancer:apoptosis β-defensin Antimicrobial

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: (A) TEM picture and size distribution of sHDL; (B)Biodistribution of DiR-labeled sHDL in mice; (C) Cellular uptake ofDiO-sHDL by SR-BI negative or positive cells without or with excessblank sHDL; (D) schematic of HDL-siRNA; (E) GPC assay of sHDL loadedwith different concentrations of PCSK9 siRNA; (F) The western blotshowed that PCSK9 siRNA-sHDL was better able to knockdown PCSK9 than thefree PCSK9 Cho-siRNA in HepG2 cells.

FIG. 2: (A) Schematic of antigens and adjuvants-loaded sHDL; (B)Addition of antigens to functional lipids containing sHDL led to theformation of lipid-antigen conjugates as measured by HPLC; (C) TheCho-CpG could be quantitatively incorporated into sHDL as measured byGPC; (D) Co-localized delivery of antigens (Ag) and adjuvants (CpG) bysHDL led to more potent cellular response than the mixture of antigensand adjuvants in montanide.

FIG. 3 shows a schematic of the synthesis ofsHDL-CSSSIINFEK(FITC)L/CpG(SEQ ID NO:386).

FIG. 4 shows homogenous particle size of sHDL-Ag/CpG as analyzed bycryoEM and dynamic light scattering.

FIGS. 5A and 5B show that compared with free antigen form, antigendelivery via sHDL significantly prolongs antigen presentation bydendritic cells.

FIG. 6 shows that sHDL-Ag/CpG significantly enhances elicitation ofantigen-specific CD8+ T cells, compared with vaccination with freeantigen mixed with conventional adjuvants.

FIG. 7 shows sHDL-Ag/CpG vaccination elicits strong CD8+ T cellresponses in tumor-bearing mice and reduces tumor growth.

FIG. 8 shows that compared with free soluble form, alpha-GalCerdelivered via sHDL significantly enhanced CD1d presentation ofantigen-presenting cells.

FIG. 9 shows that lyophilization offers a convenient method oflarge-scale synthesis of sHDL loaded with alpha-GalCer.

FIG. 10 presents a schematic of the lyophilization method for rapidpreparation of sHDL comprising encapsulated siRNA.

FIG. 11 shows a schematic of using sHDL to regulate PCSK9 for LDL-Cmanagement. As shown, (A) LDL is cleared by LDLR through endocytosis;(B) Binding of PCSK9 to LDLR leads to the degradation of LDLR inlysosomes and prevents the recycling of LDLR; (C) Knockdown of PCSK9 canupregulate LDLR and reduce LDL-C. (D) PCSK9 antibody induced by PCSK9vaccine can block the interaction between PCSK9 and LDLR, thusupregulating LDLR and reducing LDL-C.

FIG. 12: Design of sHDL nanodisc platform for “personalized” cancervaccines. a, sHDL nanodiscs, composed of phospholipids andapolipoprotein-1 mimetic peptides (22A), are engineered for co-deliveryof antigen (Ag) peptides and adjuvants. Pre-formed sHDL nanodiscsdisplaying 4 mol % DOPE-PDP (insert) are mixed with cysteine-modified Agpeptides, including tumor-associated antigens (TAAs) and tumor-specificmutated neo-antigens identified via tumor exome DNA sequencing, andsubsequent incubation with cholesterol-modified immunostimulatorymolecules (Cho-CpG) leads to formation of sHDL nanodiscs co-loaded withAg and CpG (sHDL-Ag/CpG). b, Upon administration, sHDL nanodiscsefficiently co-deliver Ag and CpG to draining lymph nodes, promotestrong and durable Ag presentation by dendritic cells (DCs) (Signal 1),and induce DC maturation (Signal 2), resulting in elicitation of robustAg-specific CD8α+ cytotoxic T lymphocyte (CTL) responses. Activated CTLsrecognize and kill their target cancer cells in peripheral tissues andexert strong anti-tumor efficacy.

FIG. 13: Effect of 22A variants and lipids on the formation of sHDLnanodisc. a, DMPC (containing 4% mol DOPE-PDP) and different 22A mutantswere used to prepare sHDL. In addition to 22A that we have usedthroughout this study, several other 22A variants, including 22Acomposed of D-amino acids, formed homogeneous sHDL nanodiscs (asanalyzed by dynamic light scattering) that remained stable up to onemonth at 4° C. N.D., not determined due to aggregation. b, Synthesis ofsHDL requires phospholipids with high transition temperature (Tm) andApoA-mimetic peptides. DPPC and DMPC (Tm=41° C. and 24° C.,respectively) but not POPC or DOPC (Tm=−2° C. and −17° C.,respectively), formed homogeneous sHDL in the presence of 22A and 4 mol% DOPE-PDP.

FIG. 14: Synthesis of functional lipid DOPE-PDP. a, DOPE, SPDP(succinimidyl 3-(2-pyridyldithio) propionate) and triethylamine (1:1:1.5molar ratio) were dissolved in chloroform and allowed to react in darkwith stirring for 5 h. b, The reaction progress was monitored by thinlayer chromatography (TLC), using the following mixture as thedeveloping solvent: chloroform/methanol/water=65/25/4 (volume ratio).c-d, The reaction mixture was purified using a silica gel column, andthe purity was assessed by c, TLC and d, HPLC using the conditiondescribed in Example VI.

FIG. 15: Preparation and characterization of sHDL-CSSSIINFEKL/CpG (SEQID NO:384), sHDL-gp100/CpG, and sHDL-Adpgk/CpG. CSSSIINFEKL (SEQ IDNO:384), CSS-gp 100 or CSS-Adpgk were incubated with sHDL-PDP, followedby insertion of Cho-CpG to sHDL-CSSSIINFEKL (SEQ ID NO:384), sHDLgp100or sHDL-Adpgk. Shown are HPLC chromatograms confirming the conjugationof a, CSSSIINFEKL (SEQ ID NO:384), c, gp 100, or e, Adpgk to sHDL-PDP.GPC of b, sHDL-CSSSIINFEKL/CpG (SEQ ID NO:384), d, sHDL-gp100/CpG, andf, sHDL-Adpgk/CpG showed homogeneity of all formulations and efficientloading of Cho-CpG in sHDL nanodiscs.

FIG. 16: Strong and durable Ag presentation mediated by sHDL nanodiscs.a, Dynamic light scattering analysis and b, transmission electronmicroscopy imaging showed uniform sHDL-Ag/CpG (10.5 nm±0.5 averagediameter) with nanodisc-like morphology. c, Homogeneity of nanodiscs wasmaintained after sterile-filtration (0.22 μm), and long-term storage (8weeks) at −20° C., followed by thawing at 37° C. d-e, BMDCs wereincubated with vaccine formulations for d, 24 h or e, indicated lengthsof time, and Ag presentation was quantified by flow-cytometry analysisof DCs stained with 25-D1.16 mAb that recognizes SIINFEKL-H-2K^(b) (SEQID NO:385) complex. f-g, Confocal microscopy images of JAWSII cells(immature DCs). f, JAWSII cells were incubated with free Ag+CpG orsHDL-Ag/CpG for 24 h and stained with 25-D1.16 mAb. Scale bars=20 μm. g,JAWSII cells were incubated with free CSSSIINFEK_((FITC))L(SEQ IDNO:386)+CpG or sHDL-CSSSIINFEK_((FITC))L(SEQ ID NO:386)/CpG for 6, 24,or 48 h, followed by staining with Hochest and Lysotracker. Scalebars=10 μm. h, BMDCs were incubated with different concentrations ofindicated formulations: low dose=20 nM SIINFEKL (SEQ ID NO:385) and 3 nMCpG; medium dose=100 nM SIINFEKL (SEQ ID NO:385) and 15 nM CpG; and highdose=500 nM SIINFEKL (SEQ ID NO:385) and 75 nM CpG. After incubation for24 h or 48 h, BMDCs were co-cultured with SIINFEKL-specific B3Z T-cellhybridoma for another 24 h, followed by assessment of T cell activation.The data show mean±SD from a representative experiment (n=3) from 2-4independent experiments. ****p<0.0001, analyzed by two-way ANOVA withTukey's HSD post-test.

FIG. 17: Strong and durable Ag presentation mediated by sHDL-Ag/CpG.BMDCs were incubated with vaccine formulations for a-b, 24 h, or c,indicated lengths of time, and Ag presentation was quantified byflow-cytometry analysis of DCs stained with 25-D1.16 mAb that recognizesSIINFEKL-H-2 Kb (SEQ ID NO:385) complex. Shown are a, the percent ofantigen presenting BMDCs at the 24 h time point, b, representativehistograms, and c, the percent of antigen presenting BMDCs over 48 h.The data show mean±SD from a representative experiment (n=3) from 2-4independent experiments. ****p<0.0001, analyzed by two-way ANOVA withTukey's HSD post-test.

FIG. 18: Ag delivery and presentation mediated by sHDL-Ag/CpG (broaderview). JAWSII cells were incubated with free CSSSIINFEK(SEQ IDNO:386)(FITC)L+CpG or sHDL-CSSSIINFEK(SEQ ID NO:386) (FITC)L/CpG for 6,24, or 48 h, and stained with Hochest and Lysotracker. Scale bar=50 μm.

FIG. 19: Intracellular delivery of sHDL (broader view). JAWSII cellswere incubated for 24 h with sHDL containing either Rhodamine-labeledDOPE (DOPE-Rhod) or Texas Red-labeled 22A and stained with Hochest andLysotracker. Scale bar=50 μm.

FIG. 20: Stimulation of bone marrow-derived dendritic cells (BMDCs) byCpG-containing formulations. BMDCs were incubated with blank sHDL or 75nM CpG formulations for 24 h. The expression levels of CD40, CD80, andCD86 were measured by flow cytometry after staining with correspondingfluorophore-labeled antibodies. The data show mean±SD from arepresentative experiment (n=3) from 3 independent experiments.

FIG. 21: Vaccine nanodiscs for LN-targeting of Ag and adjuvants andelicitation of CTL responses. a-b, C57BL/6 mice were administeredsubcutaneously at tail base with a, 31 nmol FITC-tagged Ag(CSSSIINFEK_((FITC))L(SEQ ID NO:386)) or b, 2.3 nmol Cho-CpG (20%labeled by Cy5) in free soluble or sHDL form, and fluorescence signal inthe draining inguinal LNs were quantified with IVIS after 24 h. c-f,C57BL/6 mice were immunized with the indicated formulations (15.5 nmolAg peptide and 2.3 nmol CpG) on days 0, 21, and 42. c, The frequency ofSIINFEKL-specific CD8α+ T-cells in peripheral blood was measured 7 dayspost each immunization by flow-cytometry analysis of tetramer+ CD8α+T-cells, and d, their representative scatter plots on day 49 are shown.e-f, On day 50, pre-vaccinated animals were challenged with subcutaneousflank injection of 2×10⁵ B16OVA cells. e, Tumor growth and f, overallsurvival are shown. g-h, C57BL/6 mice were immunized with the indicatedformulations in a biweekly interval. Shown are g, percent ofSIINFEKL-specific CD8α+ T-cells among PBMCs and h, ELISPOT analysis ofIFN-γ spot-forming cells among splenocytes after ex vivo restimulationwith SIINFEKL (SEQ ID NO:385) on day 42. The data show mean±SD from arepresentative experiment (n=4-5) from 2-3 independent experiments.*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, analyzed by (a-b)two-tailed unpaired Student's t test, (c,e,g) two-way ANOVA with Tukey'sHSD post-test, or (0 log-rank (Mantel-Cox) test. Asterisks in panel eindicate statistically significant differences between sHDL-Ag/CpG andSIINFEKL+CpG+Montanide.

FIG. 22: Colocalization of antigen peptides and sHDL in dLNs aftersubcutaneous administration. sHDL-CSSSIINFEK(SEQ ID NO:386) (FITC)Lnanodiscs incorporated with Cy5-labeled 22A were injected subcutaneously(31 nmol antigen peptides/mouse) at the tail base of C57BL/6 mice. After24 h, draining inguinal lymph nodes were harvested and frozen sectionswere prepared for confocal microscopy. The confocal images showedantigen peptides and 22A were colocalized in the lymph nodes (indicatedby white arrows). Scale bar=50 μm.

FIG. 23: Elicitation of CTL responses with sHDL-Ag/CpG vaccination.C57BL/6 mice were immunized with the indicated formulations in abiweekly interval. Shown are representative scatter plots forSIINFEKL-specific CD8+ T-cells among PBMCs on day 35 and their effectorCD8+ T-cell phenotype as analyzed by CD44 and CD62L staining.

FIG. 24: Therapeutic vaccination against melanoma with sHDL-Ag/CpG.C57BL/6 mice (n=5) were inoculated subcutaneously with 2×105 B16OVAcells and vaccinated on days 4 and 11 with the indicted formulations(equivalent to 15.5 nmol Ag peptide and 2.3 nmol CpG). a, Shown are thefrequency of SIINFEKL-specific CD8α+ T-cells among PBMCs as measured bytetramer staining; b, their representative scatter plots on day 17; c,B16OVA tumor growth; and d, animal survival. The data show mean±SD froma representative experiment (n=5) from 2-3 independent experiments.*p<0.05, and ****p<0.0001, analyzed by (a,c) two-way ANOVA with Tukey'sHSD post-test or (d) log-rank (Mantel-Cox) test. Asterisks in panels cindicate statistically significant differences between sHDL-Ag/CpG andall other groups.

FIG. 25: Nanodisc vaccination with tumor-associated antigens andtumor-specific neo-antigens for treatment of melanoma and colonadenocarcinoma. a-c, C57BL/6 mice were inoculated subcutaneously with2×10⁵ non-immunogenic B16F10 melanoma cells and vaccinated on days 4 and11 with the indicted formulations (equivalent to 15.5 nmol Ag peptideand 2.3 nmol CpG). a, Shown are the frequency of gp100-specific CD8α+T-cells among PBMCs; b, B16F10 tumor growth; and c, animal survival. d,Mutation of Adpgk in MC-38 murine colon adenocarcinoma cells wasconfirmed by sequencing cDNA of Adpgk. e-h, C57BL/6 mice were inoculatedsubcutaneously with 10⁵ MC-38 tumor cells and vaccinated with theindicated formulations (equivalent to 15.5 nmol mutated Adpgk peptideand 2.3 nmol CpG) on days 10, 17, and 24. Shown are e, the frequenciesof Adpgk-specific CD8α+ T-cells among PBMCs and representative scatterplots of Adpgk-tetramer+ CD8α+ T-cells on day 23; f, the percentages ofintracellular IFN-γ⁺, TNF-α⁺, and IFN-γ⁺TNF-α⁺ CD8α+ T-cells among PBMCson day 30 after ex vivo restimulation with the mutated Adpgk Ag andtheir representative scatter plots; g, growth of MC-38 tumor masses; andh, animal survival. The data show mean±SD from a representativeexperiment (n=5-8) from 2-3 independent experiments. *p<0.05, **p<0.01,***p<0.001, and ****p<0.0001, analyzed by (a,b,e,g) two-way or (f)one-way ANOVA with Tukey's HSD post-test or (c,h) log-rank (Mantel-Cox)test. Asterisks in (b,g) indicate statistically significant differencesbetween sHDL-Ag/CpG and all other groups.

FIG. 26: Therapeutic vaccination against melanoma with sHDLAg/CpG.C57BL/6 mice were inoculated subcutaneously with 2×105 B16F10 cells andvaccinated on days 4 and 11 with the indicted formulations (equivalentto 15.5 nmol Ag peptide and 2.3 nmol CpG). Shown are the representativescatter plots for gp100-specific CD8α+ T-cells among PBMCs in B16F10tumor-bearing mice on day 17.

FIGS. 27A-C: cDNA sequencing of MC-38 cells for mutated Adpgkneoantigen. Two different lengths (485 bp and 250 bp) of cDNA for theneoantigen Adpgk mRNA (amino acid sequence ASMTNMELM (SEQ ID NO:383))were prepared by using two different sets of primers. The sequence ofcDNA was analyzed by DNA sequencing. Shown are A, two different lengthsof cDNA bands on agarose gel and the results of Sanger DNA sequencingfor B, 485 bp cDNA and C, 250 bp cDNA. Arrows indicate the mutation ofG→T.

FIG. 28: Nanodisc-based vaccination with multivalent neo-antigenpeptides elicited strong CD4+ and CD8+ T cell responses. (a) PBMCs frommice vaccinated with sHDL-M30/M27/CpG showed strong IFN gamma secretionfrom CD4+ T cells upon restimulation by M30 peptide. (b) PBMCs from micevaccinated with sHDL-M30/M27/CpG showed strong IFN gamma secretion fromCD8+ T cells upon restimulation by M27 peptide. Data represent mean±SD(n=3-4).

FIG. 29: Nanoparticle formulations improve CD8+ T cell responses andtherapeutic effect of neo-antigen peptide vaccination. C57BL/6 mice wereinoculated with tumor cells (1×10⁵ MC38 cells per mouse) on the rightflank by subcutaneous injection on day 0. Mice were vaccinated on days10 and 17 with 15.5 nmol of ASMTNMELM (SEQ ID NO:383) and 2.3 nmol ofCpG in either soluble for liposomal forms. AuNP (gold nanoparticles)groups were immunized on day 10 and exposed to laser or not on day 11,followed by tetramer staining on day 17. (a) Percent of antigen specificCD8+ T cells among PBMCs elicited by different formulations on day 7post last vaccination. (b) Tumor growth curves for indicatedformulations. Data represent mean±SD (n=3-5).

FIG. 30 describes a cell culture process for recombinant production ofapolipoproteins.

FIG. 31 describes a process for the purification of apolipoproteins.

FIG. 32 describes a process for the purification of ApoA-I from plasma.

FIG. 33 describes a process for chemical synthesis ofapolipoprotein-mimetic peptides.

FIG. 34 describes a process for apolipoprotein-mimetic peptidesynthesis.

FIG. 35 describes the characterization of HDL made with full lengthApolipoprotein A-I.

FIG. 36 describes the characterization of HDL made with full lengthApolipoprotein A-I, and in particular, dynamic light scattering data ofHDL (with intensity averaged).

FIG. 37A and B describes the characterization of HDL made with fulllength Apolipoprotein A-I made by cholate/bio-bead method.

FIG. 38 describes a 22A-sHDL prepared by co-lyophilization, and analyzedwith GPC.

FIG. 39 describes a 22A-sHDL prepared by co-lyophilization, and analyzedwith DLS.

FIG. 40 describes experiments wherein C57BL/6 mice were immunized withsHDL-CpG (equivalent to 2.3 nmol CpG per dose) for 3 times in an 1-weekinterval. FIG. 40 shows the percent of 22A-specific CD4+ T cells (a),22A-specific CD8+ T cells (b) among PBMCs one week after the thirdvaccination, and (c) the titers of IgG antibody against 22A one weekafter the third vaccination. Data represent mean±SD from arepresentative experiment (n=3) from 2 independent experiments. NS,non-statistically significant.

FIG. 41: Nanodisc-based neo-antigen vaccination combined with immunecheckpoint blockade for treatment of colon adenocarcinoma. a, C57BL/6mice were inoculated subcutaneously with 10⁵ MC-38 tumor cells andvaccinated with the indicated formulations (equivalent to 15.5 nmolmutated Adpgk peptide and 2.3 nmol CpG) on days 10, 17, and 24. Shownare the percent of intracellular IFN-γ⁺, TNF-α⁺, and IFN-γ⁺TNF-α⁺ CD8α+T-cells in peripheral blood on day 30 after ex vivo restimulation withthe mutated Adpgk Ag. Average and individual MC-38 tumor growth curvesare shown with fraction of complete tumor regression (CR). b, C57BL/6mice were inoculated subcutaneously with 10⁵ MC-38 tumor cells andvaccinated with the indicated formulations (equivalent to 15.5 nmolmutated Adpgk peptide and 2.3 nmol CpG) on days 10 and 17. On days 1 and4 after each vaccination, mice were administered intraperitoneally withαPD-1 (100 μg/mouse). Average and individual MC-38 tumor growth curvesare shown. The data show mean±SD from a representative experiment(n=5-10) from 2-3 independent experiments. *p<0.05, **p<0.01,***p<0.001, and ****p<0.0001.

FIG. 42: Nanodisc-based neo-antigen vaccination combined with immunecheckpoint blockade for treatment of melanoma. a-d, C57BL/6 mice wereinoculated subcutaneously with 10⁵ melanoma B16F10 cells and vaccinatedon days 4, 11, and 18 with indicated formulations (10 nmol of eachantigen peptide and 2.3 nmol of CpG). For the combination immunotherapy,on days 1 and 4 after each vaccination, αPD-1 and αCTLA-4 (100 μg/mouseeach) were administered intraperitoneally. Shown are a, the percent ofIFN-γ⁺CD8α+ or CD4+ T cells in peripheral blood measured byintracellular cytokine staining, and b-d, average and individual B16F10tumor growth curves. The data show mean±SD from a representativeexperiment (n=5-10) from 2-3 independent experiments. *p<0.05, **p<0.01,***p<0.001, and ****p<0.0001.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used here, the term “lipids” or “lipid molecules” refer to fattysubstances that are insoluble in water and include fats, oils, waxes,and related compounds. They may be either made in the blood (endogenous)or ingested in the diet (exogenous). Lipids are essential for normalbody function and whether produced from an exogenous or endogenoussource, they must be transported and then released for use by the cells.The production, transportation and release of lipids for use by thecells is referred to as lipid metabolism. While there are severalclasses of lipids, two major classes are cholesterol and triglycerides.Cholesterol may be ingested in the diet and manufactured by the cells ofmost organs and tissues in the body, primarily in the liver. Cholesterolcan be found in its free form or, more often, combined with fatty acidsas what is called cholesterol esters. As used herein, “lipid” or “lipidmolecule” refers to any lipophilic compound. Non-limiting examples oflipid compounds include fatty acids, cholesterol, phospholipids, complexlipids, and derivatives or analogs thereof. They are usually dividedinto at least three classes: (1) “simple lipids,” which include fats andoils as well as waxes; (2) “compound lipids,” which includephospholipids and glycolipids; and (3) “derived lipids” such assteroids. Lipids or lipid molecules suitable for use in the presentinvention include both membrane-forming lipids and non-membrane-forminglipids.

As used herein the term, “lipoproteins” refer to spherical compoundsthat are structured so that water-insoluble lipids are contained in apartially water-soluble shell. Depending on the type of lipoprotein, thecontents include varying amounts of free and esterified cholesterol,triglycerides and apoproteins or apolipoproteins. There are five majortypes of lipoproteins, which differ in function and in their lipid andapoprotein content and are classified according to increasing density:(i) chylomicrons and chylomicron remnants, (ii) very low densitylipoproteins (“VLDL”), (iii) intermediate-density lipoproteins (“IDL”),(iv) low-density lipoproteins (“LDL”), and (v) high-density lipoproteins(“HDL”). Cholesterol circulates in the bloodstream as particlesassociated with lipoproteins.

As used herein, the term “HDL” or “high density lipoprotein” refers tohigh-density lipoprotein. HDL comprises a complex of lipids and proteinsin approximately equal amounts that functions as a transporter ofcholesterol in the blood. HDL is mainly synthesized in and secreted fromthe liver and epithelial cells of the small intestine. Immediately aftersecretion, HDL is in a form of a discoidal particle containingapolipoprotein A-I (also called apoA-I) and phospholipid as its majorconstituents, and also called nascent HDL. This nascent HDL receives, inblood, free cholesterol from cell membranes of peripheral cells orproduced in the hydrolysis course of other lipoproteins, and formsmature spherical HDL while holding, at its hydrophobic center,cholesterol ester converted from said cholesterol by the action of LCAT(lecithin cholesterol acyltransferase). HDL plays an extremely importantrole in a lipid metabolism process called “reverse cholesteroltransport”, which takes, in blood, cholesterol out of peripheral tissuesand transports it to the liver. High levels of HDL are associated with adecreased risk of atherosclerosis and coronary heart disease (CHD) asthe reverse cholesterol transport is considered one of the majormechanisms for HDL's prophylactic action on atherosclerosis.

As used herein, the terms “synthetic HDL,” “sHDL,” “reconstituted HDL”,or “rHDL” refer to a particle structurally analogous to native HDL,composed of a lipid or lipids in association with at least one of theproteins of HDL, preferably Apo A-I or a mimetic thereof. Typically, thecomponents of sHDL may be derived from blood, or produced by recombinanttechnology.

As used herein, the term “complexed” as used herein relates to thenon-covalent interaction of a biomacromolecule agent (e.g., antigen,adjuvant, etc) with a nanoparticle and/or microparticle.

As used herein, the term “conjugated” as used herein indicates acovalent bond association between a a biomacromolecule agent (e.g.,antigen, adjuvant, etc) and a nanoparticle and/or microparticle.

As used herein, the term “encapsulated” refers to the location of abiomacromolecule agent (e.g., antigen, adjuvant, etc) that is enclosedor completely contained within the inside of a nanoparticle and/ormicroparticle.

As used herein, the term “absorbed” refers to a biomacromolecule agent(e.g., antigen, adjuvant, etc) that is taken into and stably retained inthe interior, that is, internal to the outer surface, of a nanoparticleand/or microparticle.

As used herein, the term “adsorbed” refers to the attachment of abiomacromolecule agent (e.g., antigen, adjuvant, etc) to the externalsurface of a nanoparticle and/or microparticle. Such adsorptionpreferably occurs by electrostatic attraction. Electrostatic attractionis the attraction or bonding generated between two or more oppositelycharged or ionic chemical groups. Generally, the adsorption is typicallyreversible.

As used herein, the term “admixed” refers to a biomacromolecule agent(e.g., antigen, adjuvant, etc) that is dissolved, dispersed, orsuspended in a nanoparticle and/or microparticle. In some cases, thebiomacromolecule agent may be uniformly admixed in the nanoparticleand/or microparticle.

As used herein, the terms “biological biomacromolecule” or“biomacromolecule” or “biomacromolecule agent” as used herein refer to amolecule with a molecular mass exceeding 1 kDa which can be isolatedfrom an organism or from cellular culture, e.g., eukaryotic (e.g.,mammalian) cell culture or prokaryotic (e.g., bacterial) cell culture.In some embodiments, the use of the term refers to polymers, e.g.,biopolymers such as nucleic acids (including, but not limited to, RNA,siRNA, microRNA, interference RNA, mRNA, replicon mRNA, RNA-analogues,DNA, etc.), polypeptides (such as proteins), carbohydrates, and lipids.In some embodiments, the term “biomacromolecule” refers to a protein. Insome embodiments, the term “biomacromolecule” refers to a recombinantprotein or a fusion protein. In some embodiments, the protein issoluble. In some embodiments, the biomacromolecule is an antibody, e.g.,a monoclonal antibody. In some embodiments, the biomacromolecule is anadjuvant, an antigen, a therapeutic agent, an imaging agent, etc.

As used herein, the term “antigen” is defined herein as a molecule whichcontains one or more epitopes that will stimulate a hosts immune systemto make a cellular antigen-specific immune response, and/or a humoralantibody response. Antigens can be peptides, proteins, polysaccharides,saccharides, lipids, nucleic acids, and combinations thereof. Theantigen can be derived from a virus, bacterium, parasite, plant,protozoan, fungus, tissue or transformed cell such as a cancer orleukemic cell and can be a whole cell or immunogenic component thereof,e.g., cell wall components. An antigen may be an oligonucleotide orpolynucleotide which expresses an antigen. Antigens can be natural orsynthetic antigens, for example, haptens, polyepitopes, flankingepitopes, and other recombinant or synthetically derived antigens (see,e.g., Bergmann, et al., Eur. J. Immunol., 23:2777-2781 (1993); Bergmann,et al., J. Immunol., 157:3242-3249 (1996); Suhrbier, Immunol. and CellBiol., 75:402-408 (1997)).

As used herein, the term “neo-antigen” or “neo-antigenic” means a classof tumor antigens that arises from a tumor-specific mutation(s) whichalters the amino acid sequence of genome encoded proteins.

As used herein, the term “tumor-specific antigen” is defined herein asan antigen that is unique to tumor cells and does not occur in or onother cells in the body.

As used herein, the term “tumor-associated antigen” is defined herein asan antigen that is not unique to a tumor cell and is also expressed inor on a normal cell under conditions that fail to induce an immuneresponse to the antigen.

As used herein, the term “adjuvant” is defined herein as a substanceincreasing the immune response to other antigens when administered withother antigens. Adjuvants are also referred to herein as “immunepotentiators” and “immune modulators”.

As used herein, the term “antigen-presenting cells” are defined hereinas highly specialized cells that can process antigens and display theirpeptide fragments on the cell surface together with molecules requiredfor lymphocyte activation. The major antigen-presenting cells for Tcells are dendritic cells, macrophages and B cells. The majorantigen-presenting cells for B cells are follicular dendritic cells.

As used herein, the term “cross-presentation” is defined herein as theability of antigen-presenting cells to take up, process and presentextracellular antigens with MHC class I molecules to CD8 T cells(cytotoxic T cells). This process induces cellular immunity against mosttumors and against viruses that do not infect antigen-presenting cells.Cross-presentation is also required for induction of cytotoxic immunityby vaccination with protein antigens, for example in tumor vaccination.

As used herein, the terms “immunologic”, “immunological” or “immune”response is the development of a humoral and/or a cellular responsedirected against an antigen.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of the sHDL nanoparticles asdescribed herein (e.g., compositions comprising a sHDL nanoparticleencapsulating siRNA) (e.g., compositions comprising an sHDL nanoparticleconfigured to activate an immune responses), such delivery systemsinclude systems that allow for the storage, transport, or delivery ofsuch compositions and/or supporting materials (e.g., writteninstructions for using the materials, etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the necessary agents and/or supporting materials. As usedherein, the term “fragmented kit” refers to delivery systems comprisingtwo or more separate containers that each contain a subportion of thetotal kit components. The containers may be delivered to the intendedrecipient together or separately. For example, a first container maycontain a composition comprising an sHDL nanoparticle or the ingredientsnecessary to synthesize such an sHDL nanoparticle, while a secondcontainer contains a second agent (e.g., siRNA, an antigen, an adjuvant)(e.g., an antibiotic or spray applicator). Indeed, any delivery systemcomprising two or more separate containers that each contains asubportion of the total kit components are included in the term“fragmented kit.” In contrast, a “combined kit” refers to a deliverysystem containing all of the components necessary to synthesize andutilize any of the sHDL nanoparticles as described (e.g., in a singlebox housing each of the desired components). The term “kit” includesboth fragmented and combined kits.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

As used herein, the term “drug” or “therapeutic agent” is meant toinclude any molecule, molecular complex or substance administered to anorganism for diagnostic or therapeutic purposes, including medicalimaging, monitoring, contraceptive, cosmetic, nutraceutical,pharmaceutical and prophylactic applications. The term “drug” is furthermeant to include any such molecule, molecular complex or substance thatis chemically modified and/or operatively attached to a biologic orbiocompatible structure.

As used herein, the term “solvent” refers to a medium in which areaction is conducted. Solvents may be liquid but are not limited toliquid form. Solvent categories include but are not limited to nonpolar,polar, protic, and aprotic.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nanoparticles associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed)biomacromolecule agents configured for treating, preventing orameliorating various types of disorders, and methods of synthesizing thesame. In particular, the present invention is directed to compositionscomprising nanoparticles (e.g., synthetic high density lipoprotein(sHDL)) associated with (e.g., complexed, conjugated, encapsulated,absorbed, adsorbed, admixed) biomacromolecule agents (e.g., nucleicacid, peptides, glycolipids, etc.), methods for synthesizing suchnanoparticles, as well as systems and methods utilizing suchnanoparticles (e.g., in diagnostic and/or therapeutic settings).

Nanoparticles

The present invention is not limited to specific types or kinds ofnanoparticles associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) biomacromolecule agentsconfigured for treating, preventing or ameliorating various types ofdisorders.

Examples of nanoparticles include, but are not limited to, fullerenes(a.k.a. C₆₀, C₇₀, C₇₆, C₈₀, C₈₄), endohedral metallofullerenes (EMI's)buckyballs, which contain additional atoms, ions, or clusters insidetheir fullerene cage), trimetallic nitride templated endohedralmetallofullerenes (TNT EMEs, high-symmetry four-atom molecular clusterendohedrals, which are formed in a trimetallic nitride template withinthe carbon cage), single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods (nanotubes with internal metallo-fullerenes and/or otherinternal chemical structures), carbon nanohorns, carbon nanohornpeapods, liposomes, nanoshells, dendrimers, quantum dots,superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles.The particle embodiment can also include microparticles with thecapability to enhance effectiveness or selectivity. Other non-limitingexemplary nanoparticles include glass and polymer micro- andnano-spheres, biodegradable PLGA micro- and nano-spheres, gold, silver,carbon, and iron nanoparticles.

In some embodiments, the nanoparticle is a modified micelle. In theseembodiments, the modified micelle comprises polyol polymers modified tocontain a hydrophobic polymer block. The term “hydrophobic polymerblock” as used in the present disclosure indicates a segment of thepolymer that on its own would be hydrophobic. The term “micelle” as usedherein refers to an aggregate of molecules dispersed in a liquid. Atypical micelle in aqueous solution forms an aggregate with thehydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic single tail regions in the micelle centre.In some embodiments the head region may be, for example, a surfaceregion of the polyol polymer while the tail region may be, for example,the hydrophobic polymer block region of the polyol polymer.

The invention further encompasses use of particles on the micrometerscale in addition to the nanometer scale. Where microparticles are used,it is preferred that they are relatively small, on the order of 1-50micrometers. For ease of discussion, the use herein of “nanoparticles”encompasses true nanoparticles (sizes of from about 1 nm to about 1000nm), microparticles (e.g., from about 1 micrometer to about 50micrometers), or both.

Examples of nanoparticles include, by way of example and withoutlimitation, paramagnetic nanoparticles, superparamagnetic nanoparticles,metal nanoparticles, fullerene-like materials, inorganic nanotubes,dendrimers, dendrimers with covalently attached metal chelates,nanofibers, nanohorns, nano-onions, nanorods, nanoropes and quantumdots. In some embodiments, a nanoparticle is a metal nanoparticle (forexample, a nanoparticle of gold, palladium, platinum, silver, copper,nickel, cobalt, iridium, or an alloy of two or more thereof).Nanoparticles can include a core or a core and a shell, as in core-shellnanoparticles.

In some embodiments, the nanoparticles are sHDL nanoparticles.Generally, sHDL nanoparticles are composed of a mixture of HDLapolipoprotein and an amphipathic lipid.

The present invention is not limited to use of a particular type or kindof HDL apolipoprotein. HDL apolipoproteins include, for exampleapolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II),apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), andapolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein isselected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I,preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-IV,ApoA-V, preproApoE, proApoE, ApoE, preproApoA-IMilano, proApoA-IMilanoApoA-IMilano preproApoA-IParis , proApoA-IParis, and ApoA-IParis andpeptide mimetics of these proteins mixtures thereof. Preferably, thecarrier particles are composed of Apo A-I or Apo A-II, however the useof other lipoproteins including apolipoprotein A4, apolipoprotein Cs orapolipoprotein E may be used alone or in combination to formulatecarrier particle mixtures for delivery of therapeutic agents. In someembodiments, mimetics of such HDL apolipoproteins are used.

ApoA-I is synthesized by the liver and small intestine aspreproapolipoprotein which is secreted as a proprotein that is rapidlycleaved to generate a mature polypeptide having 243 amino acid residues.ApoA-I consists mainly of 6 to 8 different 22 amino acid repeats spacedby a linker moiety which is often proline, and in some cases consists ofa stretch made up of several residues. ApoA-I forms three types ofstable complexes with lipids: small, lipid-poor complexes referred to aspre-beta-1 HDL; flattened discoidal particles containing polar lipids(phospholipid and cholesterol) referred to as pre-beta-2 HDL; andspherical particles containing both polar and nonpolar lipids, referredto as spherical or mature HDL (HDL₃ and HDL₂). Most HDL in thecirculating population contain both ApoA-I and ApoA-II (the second majorHDL protein).

In some embodiments, ApoA-I agonists or mimetics are provided. In someembodiments, such ApoA-I mimetics are capable of forming amphipathica-helices that mimic the activity of ApoA-I, and have specificactivities approaching or exceeding that of the native molecule. Insome, the ApoA-I mimetics are peptides or peptide analogues that: formamphipathic helices (in the presence of lipids), bind lipids, formpre-β-like or HDL-like complexes, activate lecithin: cholesterolacyltransferase (LCAT), increase serum levels of HDL fractions, andpromote cholesterol efflux.

The present invention is not limited to use of a particular ApoA-Imimetic. In some embodiments, any of the ApoA-I mimetics described inSrinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 areutilized. In some embodiments, any of the ApoA-I mimetics described inU.S. Patent Application Publication Nos. 20110046056 and 20130231459 areutilized.

In some embodiments, the “22A” ApoA-I mimetic is used(PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) (see, Examples I-IV) (see, e.g.,U.S. Pat. No. 7,566,695). In some embodiments, any of the followingApoA-I mimetics shown in Table 2 as described in U.S. Pat. No. 7,566,695are utilized:

TABLE 2 ApoA-I mimetics SEQ ID NO AMINO ACID SEQUENCE (SEQ ID NO: 1)PVLDLFRELLNELLEZLKQKLK (SEQ ID NO: 2) GVLDLFRELLNELLEALKQKLKK(SEQ ID NO: 3) PVLDLFRELLNELLEWLKQKLK (SEQ ID NO: 4)PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 5) pVLDLFRELLNELLEALKQKLKK(SEQ ID NO: 6) PVLDLFRELLNEXLEALKQKLK (SEQ ID NO: 7)PVLDLFKELLNELLEALKQKLK (SEQ ID NO: 8) PVLDLFRELLNEGLEALKQKLK(SEQ ID NO: 9) PVLDLFRELGNELLEALKQKLK (SEQ ID NO: 10)PVLDLFRELLNELLEAZKQKLK (SEQ ID NO: 11) PVLDLFKELLQELLEALKQKLK(SEQ ID NO: 12) PVLDLFRELLNELLEAGKQKLK (SEQ ID NO: 13)GVLDLFRELLNEGLEALKQKLK (SEQ ID NO: 14) PVLDLFRELLNELLEALOQOLO(SEQ ID NO: 15) PVLDLFRELWNELLEALKQKLK (SEQ ID NO: 16)PVLDLLRELLNELLEALKQKLK (SEQ ID NO: 17) PVLELFKELLQELLEALKQKLK(SEQ ID NO: 18) GVLDLFRELLNELLEALKQKLK (SEQ ID NO: 19)pVLDLFRELLNEGLEALKQKLK (SEQ ID NO: 20) PVLDLFREGLNELLEALKQKLK(SEQ ID NO: 21) pVLDLFRELLNELLEALKQKLK (SEQ ID NO: 22)PVLDLFRELLNELLEGLKQKLK (SEQ ID NO: 23) PLLELFKELLQELLEALKQKLK(SEQ ID NO: 24) PVLDLFRELLNELLEALQKKLK (SEQ ID NO: 25)PVLDFFRELLNEXLEALKQKLK (SEQ ID NO: 26) PVLDLFRELLNELLELLKQKLK(SEQ ID NO: 27) PVLDLFRELLNELZEALKQKLK (SEQ ID NO: 28)PVLDLFRELLNELWEALKQKLK (SEQ ID NO: 29) AVLDLFRELLNELLEALKQKLK(SEQ ID NO: 30) PVLDLPRELLNELLEALKQKLK¹ (SEQ ID NO: 31)PVLDLFLELLNEXLEALKQKLK (SEQ ID NO: 32) XVLDLFRELLNELLEALKQKLK(SEQ ID NO: 33) PVLDLFREKLNELLEALKQKLK (SEQ ID NO: 34)PVLDZFRELLNELLEALKQKLK (SEQ ID NO: 35) PVLDWFRELLNELLEALKQKLK(SEQ ID NO: 36) PLLELLKELLQELLEALKQKLK (SEQ ID NO: 37)PVLDLFREWLNELLEALKQKLK (SEQ ID NO: 38) PVLDLFRELLNEXLEAWKQKLK(SEQ ID NO: 39) PVLDLFRELLEELLKALKKKLK (SEQ ID NO: 40)PVLDLFNELLRELLEALQKKLK (SEQ ID NO: 41) PVLDLWRELLNEXLEALKQKLK(SEQ ID NO: 42) PVLDEFREKLNEXWEALKQKLK (SEQ ID NO: 43)PVLDEFREKLWEXLEALKQKLK (SEQ ID NO: 44) pvldefreklneXlealkqklk(SEQ ID NO: 45) PVLDEFREKLNEXLEALKQKLK (SEQ ID NO: 46)PVLDLFREKLNEXLEALKQKLK (SEQ ID NO: 47) ~VLDLFRELLNEGLEALKQKLK(SEQ ID NO: 48) pvLDLFRELLNELLEALKQKLK (SEQ ID NO: 49)PVLDLFRNLLEKLLEALEQKLK (SEQ ID NO: 50) PVLDLFRELLWEXLEALKQKLK(SEQ ID NO: 51) PVLDLFWELLNEXLEALKQKLK (SEQ ID NO: 52)PVWDEFREKLNEXLEALKQKLK (SEQ ID NO: 53) VVLDLFRELLNELLEALKQKLK(SEQ ID NO: 54) PVLDLFRELLNEWLEALKQKLK (SEQ ID NO: 55)P~~~LFRELLNELLEALKQKLK (SEQ ID NO: 56) PVLDLFRELLNELLEALKQKKK(SEQ ID NO: 57) PVLDLFRNLLEELLKALEQKLK (SEQ ID NO: 58)PVLDEFREKLNEXLEALKQKL~ (SEQ ID NO: 59) LVLDLFRELLNELLEALKQKLK(SEQ ID NO: 60) PVLDLFRELLNELLEALKQ~~~ (SEQ ID NO: 61)PVLDEFRWKLNEXLEALKQKLK (SEQ ID NO: 62) PVLDEWREKLNEXLEALKQKLK(SEQ ID NO: 63) PVLDFFREKLNEXLEALKQKLK (SEQ ID NO: 64)PWLDEFREKLNEXLEALKQKLK (SEQ ID NO: 65) ~VLDEFREKLNEXLEALKQKLK(SEQ ID NO: 66) PVLDLFRNLLEELLEALQKKLK (SEQ ID NO: 67)~VLDLFRELLNELLEALKQKLK (SEQ ID NO: 68) PVLDEFRELLKEXLEALKQKLK(SEQ ID NO: 69) PVLDEFRKKLNEXLEALKQKLK (SEQ ID NO: 70)PVLDEFRELLYEXLEALKQKLK (SEQ ID NO: 71) PVLDEFREKLNELXEALKQKLK(SEQ ID NO: 72) PVLDLFRELLNEXLWALKQKLK (SEQ ID NO: 73)PVLDEFWEKLNEXLEALKQKLK (SEQ ID NO: 74) PVLDKFREKLNEXLEALKQKLK(SEQ ID NO: 75) PVLDEFREKLNEELEALKQKLK (SEQ ID NO: 76)PVLDEFRELLFEXLEALKQKLK (SEQ ID NO: 77) PVLDEFREKLNKXLEALKQKLK(SEQ ID NO: 78) PVLDEFRDKLNEXLEALKQKLK (SEQ ID NO: 79)PVLDEFRELLNELLEALKQKLK (SEQ ID NO: 80) PVLDLFERLLNELLEALQKKLK(SEQ ID NO: 81) PVLDEFREKLNWXLEALKQKLK (SEQ ID NO: 82)~~LDEFREKLNEXLEALKQKLK (SEQ ID NO: 83) PVLDEFREKLNEXLEALWQKLK(SEQ ID NO: 84) PVLDEFREKLNELLEALKQKLK (SEQ ID NO: 85)P~LDLFRELLNELLEALKQKLK (SEQ ID NO: 86) PVLELFERLLDELLNALQKKLK(SEQ ID NO: 87) pllellkellqellealkqklk (SEQ ID NO: 88)PVLDKFRELLNEXLEALKQKLK (SEQ ID NO: 89) PVLDEFREKLNEXLWALKQKLK(SEQ ID NO: 90) ~~~DEFREKLNEXLEALKQKLK (SEQ ID NO: 91)PVLDEFRELLNEXLEALKQKLK (SEQ ID NO: 92) PVLDEFRELYNEXLEALKQKLK(SEQ ID NO: 93) PVLDEFREKLNEXLKALKQKLK (SEQ ID NO: 94)PVLDEFREKLNEALEALKQKLK (SEQ ID NO: 95) PVLDLFRELLNLXLEALKQKLK(SEQ ID NO: 96) pvldlfrellneXlealkqklk (SEQ ID NO: 97)PVLDLFRELLNELLE~~~~~~~ (SEQ ID NO: 98) PVLDLFRELLNEELEALKQKLK(SEQ ID NO: 99) KLKQKLAELLENLLERFLDLVP (SEQ ID NO: 100)pvldlfrellnellealkqklk (SEQ ID NO: 101) PVLDLFRELLNWXLEALKQKLK(SEQ ID NO: 102) PVLDLFRELLNLXLEALKEKLK (SEQ ID NO: 103)PVLDEFRELLNEELEALKQKLK (SEQ ID NO: 104) P~~~~~~~LLNELLEALKQKLK(SEQ ID NO: 105) PAADAFREAANEAAEAAKQKAK (SEQ ID NO: 106)PVLDLFREKLNEELEALKQKLK (SEQ ID NO: 107) klkqklaellenllerfldlvp(SEQ ID NO: 108) PVLDLFRWLLNEXLEALKQKLK (SEQ ID NO: 109)PVLDEFREKLNERLEALKQKLK (SEQ ID NO: 110) PVLDEFREKLNEXXEALKQKLK(SEQ ID NO: 111) PVLDEFREKLWEXWEALKQKLK (SEQ ID NO: 112)PVLDEFREKLNEXSEALKQKLK (SEQ ID NO: 113) PVLDEFREKLNEPLEALKQKLK(SEQ ID NO: 114) PVLDEFREKLNEXMEALKQKLK (SEQ ID NO: 115)PKLDEFREKLNEXLEALKQKLK (SEQ ID NO: 116) PHLDEFREKLNEXLEALKQKLK(SEQ ID NO: 117) PELDEFREKLNEXLEALKQKLK (SEQ ID NO: 118)PVLDEFREKLNEXLEALEQKLK (SEQ ID NO: 119) PVLDEFREKLNEELEAXKQKLK(SEQ ID NO: 120) PVLDEFREKLNEELEXLKQKLK (SEQ ID NO: 121)PVLDEFREKLNEELEALWQKLK (SEQ ID NO: 122) PVLDEFREKLNEELEWLKQKLK(SEQ ID NO: 123) QVLDLFRELLNELLEALKQKLK (SEQ ID NO: 124)PVLDLFOELLNELLEALOQOLO (SEQ ID NO: 125) NVLDLFRELLNELLEALKQKLK(SEQ ID NO: 126) PVLDLFRELLNELGEALKQKLK (SEQ ID NO: 127)PVLDLFRELLNELLELLKQKLK (SEQ ID NO: 128) PVLDLFRELLNELLEFLKQKLK(SEQ ID NO: 129) PVLELFNDLLRELLEALQKKLK (SEQ ID NO: 130)PVLELFNDLLRELLEALKQKLK (SEQ ID NO: 131) PVLELFKELLNELLDALRQKLK(SEQ ID NO: 132) PVLDLFRELLENLLEALQKKLK (SEQ ID NO: 133)PVLELFERLLEDLLQALNKKLK (SEQ ID NO: 134) PVLELFERLLEDLLKALNOKLK(SEQ ID NO: 135) DVLDLFRELLNELLEALKQKLK (SEQ ID NO: 136)PALELFKDLLQELLEALKQKLK (SEQ ID NO: 137) PVLDLFRELLNEGLEAZKQKLK(SEQ ID NO: 138) PVLDLFRELLNEGLEWLKQKLK (SEQ ID NO: 139)PVLDLFRELWNEGLEALKQKLK (SEQ ID NO: 140) PVLDLFRELLNEGLEALOQOLO(SEQ ID NO: 141) PVLDFFRELLNEGLEALKQKLK (SEQ ID NO: 142)PVLELFRELLNEGLEALKQKLK (SEQ ID NO: 143) PVLDLFRELLNEGLEALKQKLK*(SEQ ID NO: 144) pVLELFENLLERLLDALQKKLK (SEQ ID NO: 145)GVLELFENLLERLLDALQKKLK (SEQ ID NO: 146) PVLELFENLLERLLDALQKKLK(SEQ ID NO: 147) PVLELFENLLERLFDALQKKLK (SEQ ID NO: 148)PVLELFENLLERLGDALQKKLK (SEQ ID NO: 149) PVLELFENLWERLLDALQKKLK(SEQ ID NO: 150) PLLELFENLLERLLDALQKKLK (SEQ ID NO: 151)PVLELFENLGERLLDALQKKLK (SEQ ID NO: 152) PVFELFENLLERLLDALQKKLK(SEQ ID NO: 153) AVLELFENLLERLLDALQKKLK (SEQ ID NO: 154)PVLELFENLLERGLDALQKKLK (SEQ ID NO: 155) PVLELFLNLWERLLDALQKKLK(SEQ ID NO: 156) PVLELFLNLLERLLDALQKKLK (SEQ ID NO: 157)PVLEFFENLLERLLDALQKKLK (SEQ ID NO: 158) PVLELFLNLLERLLDWLQKKLK(SEQ ID NO: 159) PVLDLFENLLERLLDALQKKLK (SEQ ID NO: 160)PVLELFENLLERLLDWLQKKLK (SEQ ID NO: 161) PVLELFENLLERLLEALQKKLK(SEQ ID NO: 162) PVLELFENWLERLLDALQKKLK (SEQ ID NO: 163)PVLELFENLLERLWDALQKKLK (SEQ ID NO: 164) PVLELFENLLERLLDAWQKKLK(SEQ ID NO: 165) PVLELFENLLERLLDLLQKKLK (SEQ ID NO: 166)PVLELFLNLLEKLLDALQKKLK (SEQ ID NO: 167) PVLELFENGLERLLDALQKKLK(SEQ ID NO: 168) PVLELFEQLLEKLLDALQKKLK (SEQ ID NO: 169)PVLELFENLLEKLLDALQKKLK (SEQ ID NO: 170) PVLELFENLLEOLLDALQOOLO(SEQ ID NO: 171) PVLELFENLLEKLLDLLQKKLK (SEQ ID NO: 172)PVLELFLNLLERLGDALQKKLK (SEQ ID NO: 173) PVLDLFDNLLDRLLDLLNKKLK(SEQ ID NO: 174) pvlelfenllerlldalqkklk (SEQ ID NO: 175)PVLELFENLLERLLELLNKKLK (SEQ ID NO: 176) PVLELWENLLERLLDALQKKLK(SEQ ID NO: 177) GVLELFLNLLERLLDALQKKLK (SEQ ID NO: 178)PVLELFDNLLEKLLEALQKKLR (SEQ ID NO: 179) PVLELFDNLLERLLDALQKKLK(SEQ ID NO: 180) PVLELFDNLLDKLLDALQKKLR (SEQ ID NO: 181)PVLELFENLLERWLDALQKKLK (SEQ ID NO: 182) PVLELFENLLEKLLEALQKKLK(SEQ ID NO: 183) PLLELFENLLEKLLDALQKKLK (SEQ ID NO: 184)PVLELFLNLLERLLDAWQKKLK (SEQ ID NO: 185) PVLELFENLLERLLDALQOOLO(SEQ ID NO: 186) PVLELFEQLLERLLDALQKKLK (SEQ ID NO: 187)PVLELFENLLERLLDALNKKLK (SEQ ID NO: 188) PVLELFENLLDRLLDALQKKLK(SEQ ID NO: 189) DVLELFENLLERLLDALQKKLK (SEQ ID NO: 190)PVLEFWDNLLDKLLDALQKKLR (SEQ ID NO: 191) PVLDLLRELLEELKQKLK*(SEQ ID NO: 192) PVLDLFKELLEELKQKLK* (SEQ ID NO: 193)PVLDLFRELLEELKQKLK* (SEQ ID NO: 194) PVLELFRELLEELKQKLK*(SEQ ID NO: 195) PVLELFKELLEELKQKLK* (SEQ ID NO: 196)PVLDLFRELLEELKNKLK* (SEQ ID NO: 197) PLLDLFRELLEELKQKLK*(SEQ ID NO: 198) GVLDLFRELLEELKQKLK* (SEQ ID NO: 199)PVLDLFRELWEELKQKLK* (SEQ ID NO: 200) NVLDLFRELLEELKQKLK*(SEQ ID NO: 201) PLLDLFKELLEELKQKLK* (SEQ ID NO: 202)PALELFKDLLEELRQKLR* (SEQ ID NO: 203) AVLDLFRELLEELKQKLK*(SEQ ID NO: 204) PVLDFFRELLEELKQKLK* (SEQ ID NO: 205)PVLDLFREWLEELKQKLK* (SEQ ID NO: 206) PLLELLKELLEELKQKLK*(SEQ ID NO: 207) PVLELLKELLEELKQKLK* (SEQ ID NO: 208)PALELFKDLLEELRQRLK* (SEQ ID NO: 209) PVLDLFRELLNELLQKLK (SEQ ID NO: 210)PVLDLFRELLEELKQKLK (SEQ ID NO: 211) PVLDLFRELLEELOQOLO* (SEQ ID NO: 212)PVLDLFOELLEELOQOLK* (SEQ ID NO: 213) PALELFKDLLEEFRQRLK*(SEQ ID NO: 214) pVLDLFRELLEELKQKLK* (SEQ ID NO: 215)PVLDLFRELLEEWKQKLK* (SEQ ID NO: 216) PVLELFKELLEELKQKLK (SEQ ID NO: 217)PVLDLFRELLELLKQKLK (SEQ ID NO: 218) PVLDLFRELLNELLQKLK* (SEQ ID NO: 219)PVLDLFRELLNELWQKLK (SEQ ID NO: 220) PVLDLFRELLEELQKKLK (SEQ ID NO: 221)DVLDLFRELLEELKQKLK* (SEQ ID NO: 222) PVLDAFRELLEALLQLKK (SEQ ID NO: 223)PVLDAFRELLEALAQLKK (SEQ ID NO: 224) PVLDLFREGWEELKQKLK (SEQ ID NO: 225)PVLDAFRELAEALAQLKK (SEQ ID NO: 226) PVLDAFRELGEALLQLKK (SEQ ID NO: 227)PVLDLFRELGEELKQKLK* (SEQ ID NO: 228) PVLDLFREGLEELKQKLK*(SEQ ID NO: 229) PVLDLFRELLEEGKQKLK* (SEQ ID NO: 230) PVLELFERLLEDLQKKLK(SEQ ID NO: 231) PVLDLFRELLEKLEQKLK (SEQ ID NO: 232) PLLELFKELLEELKQKLK*(SEQ ID NO: 233) LDDLLQKWAEAFNQLLKK (SEQ ID NO: 234) EWLKAFYEKVLEKLKELF*(SEQ ID NO: 235) EWLEAFYKKVLEKLKELF* (SEQ ID NO: 236)DWLKAFYDKVAEKLKEAF* (SEQ ID NO: 237) DWFKAFYDKVFEKFKEFF (SEQ ID NO: 238)GIKKFLGSIWKFIKAFVG (SEQ ID NO: 239) DWFKAFYDKVAEKFKEAF (SEQ ID NO: 240)DWLKAFYDKVAEKLKEAF (SEQ ID NO: 241) DWLKAFYDKVFEKFKEFF (SEQ ID NO: 242)EWLEAFYKKVLEKLKELP (SEQ ID NO: 243) DWFKAFYDKFFEKFKEFF (SEQ ID NO: 244)EWLKAFYEKVLEKLKELF (SEQ ID NO: 245) EWLKAEYEKVEEKLKELF* (SEQ ID NO: 246)EWLKAEYEKVLEKLKELF* (SEQ ID NO: 247) EWLKAFYKKVLEKLKELF*(SEQ ID NO: 248) PVLDLFRELLEQKLK* (SEQ ID NO: 249) PVLDLFRELLEELKQK*(SEQ ID NO: 250) PVLDLFRELLEKLKQK* (SEQ ID NO: 251) PVLDLFRELLEKLQK*(SEQ ID NO: 252) PVLDLFRELLEALKQK* (SEQ ID NO: 253) PVLDLFENLLERLKQK*(SEQ ID NO: 254) PVLDLFRELLNELKQK* *indicates peptides that areN-terminal acetylated and C-terminal amidated; indicates peptides thatare N-terminal dansylated; sp indicates peptides that exhibitedsolubility problems under the experimental conditions; X is Aib, Z isNal; O is Om, He (%) designates percent helicity; mics designatesmicelles; and ~ indicates deleted amino acids.

In some embodiments, an ApoA-I mimetic having the following sequence asdescribed in U.S. Pat. No. 6,743,778 is utilized: Asp Trp Leu Lys AlaPhe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO:255).

In some embodiments, any of the following ApoA-I mimetics shown in Table3 as described in U.S. Patent Application Publication No. 2003/0171277are utilized:

TABLE 3 SEQ ID NO AMINO ACID SEQUENCE (SEQ ID NO: 256)D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO: 257)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 258)Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 259)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 260)Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 261)Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 262)Ac-D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 263)Ac-D-W-F-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 264)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 265)Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 266)Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 267)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 268)Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 269)Ac-E-W-L-K-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 270)Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 271)Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 272)Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 273)Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 274)Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 275)Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 276)AC-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 277)Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 278)Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 279)Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 280)Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 281)Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 282)Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 283)Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 284)Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 285)Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 286)Ac-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-NH₂ (SEQ ID NO: 287)Ac-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 288)Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 289)Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 290)Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ (SEQ ID NO: 291)Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 292)Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 293)Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 294)Ac-D-W-L-K-A-L-Y-D-K-V-A-E-K-L-K-E-A-L-NH₂ (SEQ ID NO: 295)Ac-D-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 296)Ac-D-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 297)Ac-E-W-L-K-A-L-Y-E-K-V-A-E-K-L-K-E-A-L-NH₂ (SEQ ID NO: 298)Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 299)Ac-E-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH₂ (SEQ ID NO: 300)Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 301)Ac-E-W-L-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 302)Ac-E-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH₂ (SEQ ID NO: 303)Ac-D-F-L-K-A-W-Y-D-K-V-A-E-K-L-K-E-A-W-NH₂ (SEQ ID NO: 304)Ac-E-F-L-K-A-W-Y-E-K-V-A-E-K-L-K-E-A-W-NH₂ (SEQ ID NO: 305)Ac-D-F-W-K-A-W-Y-D-K-V-A-E-K-L-K-E-W-W-NH₂ (SEQ ID NO: 306)Ac-E-F-W-K-A-W-Y-E-K-V-A-E-K-L-K-E-W-W-NH₂ (SEQ ID NO: 307)Ac-D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-NH₂ (SEQ ID NO: 308)Ac-D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-NH₂ (SEQ ID NO: 309)Ac-E-K-L-K-A-F-Y-E-K-V-F-E-W-A-K-E-A-F-NH₂ (SEQ ID NO: 310)Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH₂ (SEQ ID NO: 311)Ac-D-W-L-K-A-F-V-D-K-F-A-E-K-F-K-E-A-Y-NH₂ (SEQ ID NO: 312)Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH₂ (SEQ ID NO: 313)Ac-D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-NH₂ (SEQ ID NO: 314)Ac-E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F-NH₂ (SEQ ID NO: 315)Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 316)Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 317)Ac-D-W-L-K-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 318)Ac-E-W-L-K-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 319)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH₂ (SEQ ID NO: 320)Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH₂ (SEQ ID NO: 321)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH₂ (SEQ ID NO: 322)Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH₂ (SEQ ID NO: 323)Ac-D-W-L-K-A-F-Y-D-R-V-A-E-R-L-K-E-A-F-NH₂ (SEQ ID NO: 324)Ac-E-W-L-K-A-F-Y-E-R-V-A-E-R-L-K-E-A-F-NH₂ (SEQ ID NO: 325)Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH₂ (SEQ ID NO: 326)Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH₂ (SEQ ID NO: 327)Ac-D-W-L-R-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 328)Ac-E-W-L-R-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH₂ (SEQ ID NO: 329)Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-R-E-A-F-NH₂ (SEQ ID NO: 330)Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-R-E-A-F-NH₂ (SEQ ID NO: 331)Ac-D-W-L-R-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH₂ (SEQ ID NO: 332)Ac-E-W-L-R-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH₂

In some embodiments, an ApoA-I mimetic having the following sequence asdescribed in U.S. Patent Application Publication No. 2006/0069030 isutilized:

(SEQ ID NO: 333) F-A-E-K-F-K-E-A-V-K-D-Y-F-A-K-F-W-D.

In some embodiments, an ApoA-I mimetic having the following sequence asdescribed in U.S. Patent Application Publication No. 2009/0081293 isutilized:

(SEQ ID NO: 334) DWFKAFYDKVAEKFKEAF; (SEQ ID NO: 335)DWLKAFYDKVAEKLKEAF; (SEQ ID NO: 336) PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ.

In some embodiments, an ApoA-I mimetic having one of the followingsequences is utilized:

(SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF, (SEQ ID NO: 342)LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343) PVTOEFWDNLEKETEGLROEMS,(SEQ ID NO: 344) KDLEEVKAKVQ, (SEQ ID NO: 345) KDLEEVKAKVO,(SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS,(SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.

Amphipathic lipids include, for example, any lipid molecule which hasboth a hydrophobic and a hydrophilic moiety. Examples includephospholipids or glycolipids. Examples of phospholipids which may beused in the sHDL-TA nanoparticles include but are not limited todipalmitoylphosphatidylcholine (DPPC),dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof. In some embodiments,the phospholipid is complexed with an imaging agent (e.g., rhodamine(Rhod)-labeled DOPE (DOPE-Rhod)). In some embodiments, the phospholipidsare thiol reactive phospholipids such as, for example,Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP),1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanol, orN-4-(p-maleimidophenyl)butyryl) dipalmitoylphosphatidylethanolamine(MPB-DPPE)).

In some embodiments, exemplary phospholipids include, but are notlimited to, small alkyl chain phospholipids, egg phosphatidylcholine,soybean phosphatidylcholine, dipalmitoylphosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerolssuch as dimyristoylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid,dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoylsphingomyelin, phytosphingomyelin, dipalmitoylsphingomyelin,distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives. Phospholipid fractionsincluding SM and palmitoylsphingomyelin can optionally include smallquantities of any type of lipid, including but not limited tolysophospholipids, sphingomyelins other than palmitoylsphingomyelin,galactocerebroside, gangliosides, cerebrosides, glycerides,triglycerides, and cholesterol and its derivatives.

In some embodiments, the sHDL nanoparticles have a molar ratio ofphospholipid/HDL apolipoprotein from 2 to 250 (e.g., 10 to 200, 20 to100, 20 to 50, 30 to 40).

Generally, the sHDL nanoparticles so formed are spherical and have adiameter of from about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, thesHDL nanoparticles are subjected to size exclusion chromatography toyield a more homogeneous preparation.

The present invention addresses the need for improved stable andtargeted delivery (e.g., in vitro or in vivo) of biomacromolcules (e.g.,peptides, nucleic acids, glycolipids). Indeed, the present inventionaddresses such needs through providing synthetic high densitylipoprotein (sHDL) nanoparticles for stable and targeted delivery ofbiomacromolecules, including peptides, nucleic acids, and glycolipids.

Compared to other strategies, including conventional nanoparticlevehicles, sHDL nanoparticles have impressive biocompatibility andcapacity for cargo loading. For example, the ultrasmall but tunable size(e.g., 10-20 nm) enables the sHDL nanoparticles to effectively drain tolymph nodes and deliver cargo peptide antigens and nucleic acid-basedadjuvants to lymph node-resident dendritic cells, thus positioning themas an efficient platform for co-delivery of antigen and adjuvant fortumor immunotherapy. In addition, experiments conducted during thecourse of developing embodiments for the present invention demonstratedbroad applicability of the sHDL-based approach by (1) targeting deliveryof siRNA to hepatocytes, which are the natural target cells ofendogenous HDL, and (2) targeting immunostimulatory glycolid(alpha-galactosyl ceramide) to antigen presenting cells.

Such experiments further demonstrated the engineering of sHDLnanoparticles prepared with phospholipids and Apolipoprotein A-I mimeticpeptides and loaded with biomacromolecular drugs. To load peptide drugson HDL nanodiscs, synthesized thiol-reactive phospholipids were utilizedthat allowed reduction-sensitive linkage of peptides on the surfaces ofHDL nanodiscs. To load nucleic acids (including CpG motifs and siRNA),nucleic acids were modified with a cholesteryl moiety, which was shownto allow facile insertion of nucleic acids into the sHDL nanoparticles.To load glycolipids into HDL, hydrophobic interactions betweenglycolipids and HDL were utilized. Such experiments further demonstratedstable delivery of such cargo to target tissues in vitro and in vivo.

RNA Interference

In certain embodiments, the sHDL nanoparticles are used within RNAinterference methods and systems.

RNA interference is a highly conserved mechanism triggered bydouble-stranded RNA (dsRNA) and able to down regulate transcript ofgenes homologous to the dsRNA. The dsRNA is first processed by Dicerinto short duplexes of 21-23 nt, called short interfering RNAs (siRNAs).Incorporated in RNA-induced silencing complex (RISC), they are able tomediate gene silencing through cleavage of the target mRNA. “siRNA” or“small-interfering ribonucleic acid” refers to two strands ofribonucleotides which hybridize along a complementary region underphysiological conditions. The siRNA molecules comprise a double-strandedregion which is substantially identical to a region of the mRNA of thetarget gene. A region with 100% identity to the corresponding sequenceof the target gene is suitable. This state is referred to as “fullycomplementary”. However, the region may also contain one, two or threemismatches as compared to the corresponding region of the target gene,depending on the length of the region of the mRNA that is targeted, andas such may be not fully complementary. Methods to analyze and identifysiRNAs with sufficient sequence identity in order to effectively inhibitexpression of a specific target sequence are known in the art. Asuitable mRNA target region would be the coding region. Also suitableare untranslated regions, such as the 5′-UTR, the 3′-UTR, and splicejunctions as long as the regions are unique to the mRNA target and notdirected to a mRNA poly A tail.

In some embodiments, siRNA encapsulated within sHDL nanoparticles areutilized conducting methods and systems involving RNA interference.

Such embodiments are not limited to a particular size or type of siRNAmolecule. The length of the region of the siRNA complementary to thetarget, for example, may be from 15 to 100 nucleotides, 18 to 25nucleotides, 20 to 23 nucleotides, or more than 15, 16, 17 or 18nucleotides. Where there are mismatches to the corresponding targetregion, the length of the complementary region is generally required tobe somewhat longer.

In certain embodiments, it is contemplated that the siRNA deliveryapproach using sHDL nanoparticles disclosed herein (e.g., throughencapsulation of the siRNA within an sHDL nanoparticle) can be used toinhibit any gene of interest.

The present invention is not limited to particular methods forgenerating sHDL nanoparticles comprising encapsulated siRNA molecules.For example, in some embodiments, lyophilization methods are used forthe preparation of homogenous sHDL. In some embodiments, phospholipidsand ApoA mimetic peptides are dissolved in glacial acetic acid andlyophilized. In some embodiments, loading of an siRNA molecule into thesHDL nanoparticle is facilitated through cholesterol modification of thesiRNA molecule. For example, the siRNA is modified with cholesterol atthe 3′ sense strand (e.g., Kuwahara, H.; et al., Molecular Therapy 2011,19 (12), 2213-2221) and an intermediate level of chemical modificationwill be used to stabilize siRNA in the serum without significantlycompromising its silencing effect (see, e.g., Behlke, M. A.,Oligonucleotides 2008, 18 (4), 305-319). In some embodiments, thelyophilized phospholipids and ApoA mimetic peptides are hydrated (e.g.,hydrated in PBS (pH 7.4)) and thermocycled above and below thetransition temperature (Tm) of phospholipids to form blank sHDL, whichare next incubated with the cholesterol modified siRNA at roomtemperature for an optimal amount of time (e.g., 5, 10, 20, 25, 30, 35,50, 80, 120, 360 minutes) to form sHDL comprising encapsulated siRNA.FIG. 10 presents a schematic of the lyophilization method for rapidpreparation of sHDL comprising encapsulated siRNA.

Such embodiments are not limited to a particular manner ofcharacterizing the sHDL comprising encapsulated siRNA. In someembodiments, the morphology of sHDL is observed by TEM. In someembodiments, the size distribution of sHDL is analyzed by dynamic lightscattering (DLS) using a Malven Nanosizer instrument and GPC assay.

Such embodiments are not limited to a particular manner of assessing thedelivery profile of the siRNA in vitro and in vivo. In some embodiments,labelling the siRNA molecules with an imaging agent (e.g., fluorescentdye Cy3) permits visualization of the biodistribution of siRNA moleculesat the organ level and also the intracellular delivery profile. In someembodiments, RT-PCR and western blot are used to analyze the targetprotein at the mRNA level and protein level, respectively.

As such, in certain embodiments, the present invention provides methodsfor inhibiting a target gene in a cell comprising introducing into thecell a siRNA capable of inhibiting the target gene by RNA interference,wherein the siRNA comprises two RNA strands that are complementary toeach other, wherein the siRNA is encapsulated within a sHDLnanoparticle. In some embodiments, the siRNA is modified withcholesterol at the 3′ sense strand. In some embodiments, the cell iswithin a human being.

In certain embodiments, sHDL nanoparticles are provided wherein siRNAsspecific for proprotein convertase subtilisin/kexin 9 (PCSK9) areencapsulated within the sHDL nanoparticle. Compelling evidence has shownthat an elevated plasma level of low-density lipoprotein cholesterol(LDL-C) is a cardinal risk factor for coronary heart disease (CHD) (see,e.g., Law, M. R.; et al., British Medical Journal 2003, 326 (7404),1423-1427; Boekholdt, S. M.; et al., Jama-Journal of the AmericanMedical Association 2012, 307 (12), 1302-1309; Sniderman, A. D.; et al.,Circulation-Cardiovascular Quality and Outcomes 2011, 4 (3), 337-U144).PCSK9 synthesized in the liver performs important roles in regulatingLDL-C: PCSK9 can bind to the LDL receptor (LDLR) on hepatocytes andprevent the recycling of LDLR from lysosomes to the cell surface, andthis in turn leads to the down-regulation of LDLR and increased levelsof LDL-C (see, e.g., Maxwell, K. N.; et al., Proceedings of the NationalAcademy of Sciences of the United States of America 2004, 101 (18),7100-7105; Dadu, R. T.; et al., Nature Reviews Cardiology 2014, 11 (10),563-575; Horton, J. D.; et al., Trends in Biochemical Sciences 2007, 32(2), 71-77). Therefore PCSK9 inhibition can potentially decrease LDL-C(see, e.g., Shen, L.; et al., Pharmacological Research 2013, 73, 27-34).Therapeutic approaches under development for PCSK9 inhibition in vivoinclude siRNA-mediated knockdown of PCSK9 and vaccination against PCSK9(see, e.g., Fitzgerald, K.; et al., Lancet 2014, 383 (9911), 60-68;Galabova, G.; et al., Circulation 2013, 128 (22)), but both strategiesface the major challenge: how to efficiently deliver therapeutic agentsto the target cells, namely hepatocytes and immune cells, respectively,in order to maximize the in vivo efficacy of each strategy.

Previously, PCSK9 siRNA has been delivered to the hepatocytes by lipidnanoparticles (see, e.g., Frank-Kamenetsky, M.; et al., Proceedings ofthe National Academy of Sciences of the United States of America 2008,105 (33), 11915-11920) or by conjugating siRNA to N-acetylgalactosamine(GalNAc) ligands (see, e.g., Akinc, A.; et al., Molecular Therapy 2010,18 (7), 1357-1364), which allow siRNA to be targeted to hepatocytespassively or through the recognition of Asialoglycoprotein Receptor(ASGPR) on hepatocytes. However, these conventional delivery strategiescan subject the siRNA molecules to the intracellular endosomes/lysosomespathway, in which siRNA cargo can be degraded, leading to suboptimalknockdown of PCSK9. Therefore, developing strategies that can bothtarget the hepatocyte and bypass the endosomes/lysosomes pathway areurgently needed.

Use of sHDL nanoparticles comprising encapsulated PCSK9 siRNA moleculesovercomes such limitations. Indeed, sHDL nanoparticles have similarproperties to endogenous HDL, which can intrinsically target hepatocytesafter i.v. injection, thus permitting direct delivery of siRNA cargoesto the cytosol of hepatocytes and knockdown of PCSK9 without goingthrough the intracellular endosome/lysosome pathway.

In certain embodiments, sHDL comprising encapsulated PCSK9 siRNAmolecules are delivered into the cytosol where they can associate withRNA-induced silencing complex (RISC) to knockdown the PCSK9 proteins(see, e.g., Chendrimada, T. P.; et al., Nature 2005, 436 (7051),740-744; Matranga, C.; et al., Cell 2005, 123 (4), 607-620) within SR-BIexpressing hepatocytes (see, e.g., Goldstein, J. L.; Brown, M. S.,Arteriosclerosis Thrombosis and Vascular Biology 2009, 29 (4), 431-438;Wolfrum, C.; et al., Nature Biotechnology 2007, 25 (10), 1149-1157).

FIG. 11 shows a schematic of using sHDL to regulate PCSK9 for LDL-Cmanagement.

The present invention is not limited to use of a particular PCSK9 siRNAsequence. In some embodiments, the PCSK9 siRNA sequence iscross-reactive to murine, rat, nonhuman primate and human PCSK9 mRNA(see, e.g., Frank-Kamenetsky, et al., Proceedings of the NationalAcademy of Sciences of the United States of America 2008, 105 (33),11915-11920).

In certain embodiments, the present invention provides methods forinhibiting a PCSK9 gene in a cell comprising introducing into the cell aPCSK9 siRNA capable of inhibiting the PCSK9 gene by RNA interference,wherein the PCSK9 siRNA comprises two RNA strands that are complementaryto each other, wherein the PCSK9 siRNA is encapsulated within a sHDLnanoparticle. In some embodiments, the PCSK9 siRNA is modified withcholesterol at the 3′ sense strand. In some embodiments, the cell iswithin a human being.

In certain embodiments, the present invention provides methods forreducing serum LDL-C levels in patient (e.g., human patient), comprisingadministering to the patient a therapeutically effective amount of apharmaceutical composition comprising a PCSK9 siRNA encapsulated withina sHDL nanoparticle, wherein the PCSK9 siRNA is capable of inhibitingthe PCSK9 gene by RNA interference, wherein the PCSK9 siRNA comprisestwo RNA strands that are complementary to each other, wherein inhibitingof the PCSK9 gene results in reduction of serum LDL-C levels.

In certain embodiments, the present invention provides methods fortreating coronary heart disease in a patient through reducing serumLDL-C levels in the patient, comprising administering to the patient atherapeutically effective amount of a pharmaceutical compositioncomprising a PCSK9 siRNA encapsulated within a sHDL nanoparticle,wherein the PCSK9 siRNA is capable of inhibiting the PCSK9 gene by RNAinterference, wherein the PCSK9 siRNA comprises two RNA strands that arecomplementary to each other, wherein inhibiting of the PCSK9 generesults in reduction of serum LDL-C levels.

In certain embodiments, the sHDL nanoparticles are used to activate animmune response. Such embodiments are not limited to a particular mannerof activating an immune response.

Biomacromolecule Delivery

In certain embodiments, the present invention provides compositionscomprising a nanoparticle (as described herein), wherein any kind ofbiomacromolecule agent (e.g., nucleic acid, peptides, glycolipids, etc.)is associated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the nanoparticle.

In some embodiments, the biomacromolecule agent is a peptide.

For example, in some embodiments, the peptide is an antigen. In someembodiments wherein the peptide is an antigen, the composition furthercomprises an adjuvant (as described herein).

As noted, the peptide is not limited to a particular type of peptide.

In some embodiments, the peptide is Adrenocorticotropic Hormone (ACTH),a growth hormone peptide, a Melanocyte Stimulating Hormone (MSH),Oxytocin, Vasopressin, Corticotropin Releasing Factor (CRF), aCRF-related peptide, a Gonadotropin Releasing Hormone Associated Peptide(GAP), Growth Hormone Releasing Factor (GRF), Lutenizing Hormone ReleaseHormone (LH-RH), an orexin, a Prolactin Releasing Peptide (PRP), asomatostatin, Thyrotropin Releasing Hormone (THR), a THR analog,Calcitonin (CT), a CT-precursor peptide, a Calcitonin Gene RelatedPeptide (CGRP), a Parathyroid Hormone (PTH), a Parathyroid HormoneRelated Protein (PTHrP), Amylin, Glucagon, Insulin, an Insulin-likepeptide, NeuroPeptide Y (NPY), a Pancreatic Polypeptide (PP), Peptide YY(PYY), Cholecystokinin (CCK), a CCK-related peptide, Gastrin ReleasingPeptide (GRP), Gastrin, a Gastrin-related peptide, a Gastrin inhibitorypeptide, Motilin, Secretin, Vasoactive Intestinal Peptide (VIP), aVIP-related peptide, an Atrial-Natriuretic Peptide (ANP), a BrainNatriuretic Peptide (BNP), a C-Type Natriuretic Peptide(CNP), atachykinin, an angiotensin, a renin substrate, a renin inhibitor, anendothelin, an endothelin-related peptide, an opioid peptide, a thymicpeptide, an adrenomedullin peptide, an allostatin peptide, an amyloidbeta-protein fragment, an antimicrobial peptide, an antioxidant peptide,an apoptosis related peptide, a Bag Cell Peptide (BCPs), Bombesin, abone Gla protein peptide, a Cocaine and Amphetamine Related Transcript(CART) peptide, a cell adhesion peptide, a chemotactic peptide, acomplement inhibitor, a cortistatin peptide, a fibronectin fragment, afibrin related peptide, FMRF, a FMRF amide-related peptide (FaRP),Galanin, a Galanin-related peptide, a growth factor, a growthfactor-related peptide, a G-Therapeutic Peptide-Binding Proteinfragment, Gualylin, Uroguanylin, an Inhibin peptide, Interleukin (IL),an Interleukin Receptor protein, a laminin fragment, a leptin fragmentpeptide, a leucokinin, Pituitary Adenylate Cyclase ActivatingPolypeptide (PAPCAP), Pancreastatin, a polypeptide repetitive chain, asignal transducing reagent, a thrombin inhibitor, a toxin, a trypsininhibitor, a virus-related peptide, an adjuvant peptide analog, AlphaMating Factor, Antiarrhythmic Peptide, Anorexigenic Peptide, Alpha-1Antitrypsin, Bovine Pineal Antireproductive Peptide, Bursin, C3 PeptideP16, Cadherin Peptide, Chromogranin A Fragment, ContraceptiveTetrapeptide, Conantokin G, Conantokin T, Crustacean CardioactivePeptide, C-Telopeptide, Cytochrome b588 Peptide, Decorsin, DeliciousPeptide, Delta-Sleep-Inducing Peptide, Diazempam-Binding InhibitorFragment, Nitric Oxide Synthase Blocking Peptide, OVA Peptide, PlateletCalpain Inhibitor (P1), Plasminogen Activator Inhibitor 1, Rigin,Schizophrenia Related Peptide, Sodium Potassium Atherapeutic PeptidaseInhibitor-1, Speract, Sperm Activating Peptide, Systemin, a Thrombinreceptor agonist, Tuftsin, Adipokinetic Hormone, Uremic Pentapeptide,Antifreeze Polypeptide, Tumor Necrosis Factor (TNF), Leech [DesAsp10]Decorsin, L-Omithyltaurine Hydrochloride, P-AminophenylacetylTuftsin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro,Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly,D-Ala-Leu, D-D-D-D-D, D-D-D-D-D-D, N-P-N-A-N-P-N-A, V-A-I-T-V-L-V-K,V-G-V-R-V-R, V-I-H-S, V-P-D-P-R, Val-Thr-Cys-Gly, R-S-R, Sea UrchinSperm Activating Peptide, a SHU-9119 antagonist, a MC3-R antagonist, aMC4-R antagonist, Glaspimod, HP-228, Alpha 2-Plasmin Inhibitor, APCTumor Suppressor, Early Pregnancy Factor, Gamma Interferon, GlandularKallikrei N-1, Placental Ribonuclease Inhibitor, Sarcolecin BindingProtein, Surfactant Protein D, Wilms' Tumor Suppressor, GABAB 1bReceptor Peptide, Prion Related Peptide (iPRP13), Choline BindingProtein Fragment, Telomerase Inhibitor, Cardiostatin Peptide, EndostatinDerived Peptide, Prion Inhibiting Peptide, N-Methyl D-Aspartate ReceptorAntagonist, and C-PeptideAnalog.

In some embodiments, the peptide is selected from177Lu-DOTA0-Tyr3-Octreotate, Abarelix acetate, ADH-1, Afamelanotidec,melanotan-1, CUV1647, Albiglutide, Aprotinin, Argipressin, Atosibanacetate, Bacitracin, Bentiromide, a BH3 domain, Bivalirudin, Bivalirudintrifluoroacetate hydrate, Blisibimod, Bortezomib, Buserelin, Buserelinacetate, Calcitonin, Carbetocin, Carbetocin acetate, Cecropin A and B,Ceruletide, Ceruletide diethylamine, Cetrorelix, Cetrorelix acetate,Ciclosporine, Cilengitidec, EMD121974, Corticorelin acetate injection,hCRF, Corticorelin ovine triflutate, corticorelin trifluoroacetate,Corticotropin, Cosyntropin, ACTH 1-24, tetracosactide hexaacetate,Dalbavancin, Daptomycin, Degarelix acetate, Depreotide trifluoroacetate(plus sodium pertechnetate), Desmopressin acetate, Desmopressin DDAVP,Dulaglutide, Ecallantide, Edotreotide (plus yttrium-90), Elcatoninacetate, Enalapril maleate (or 2-butanedioate), Enfuvirtide,Eptifibatide, Exenatide, Ganirelix acetate, Glatiramer acetate,Glutathion, Gonadorelin, Gonadorelin acetate, GnRH, LHRH, Goserelin,Goserelin acetate, Gramicidin, Histrelin acetate, Human calcitonin,Icatibant, Icatibant acetate, IM862, oglufanide disodium, KLAKLAK,Lanreotide acetate, Lepirudin, Leuprolide, Leuprolide acetate,leuprorelin, Liraglutide, Lisinopril, Lixisenatide, Lypressin,Magainin2, MALP-2Sc, macrophage-activating lipopeptide-2 synthetic,Nafarelin acetate, Nesiritide, NGR-hTNF, Octreotide acetate,Oritavancin, Oxytocin, Pasireotide, Peginesatide, Pentagastrin,Pentetreotide (plus indium-111), Phenypressin, Pleurocidin, Pramlintide,Protirelin, thyroliberin, TRH, TRF, Salmon calcitonin, Saralasinacetate, Secretin (human), Secretin (porcine), Semaglutide, Seractideacetate, ACTH, corticotropin, Sermorelin acetate, GRF 1-29, Sinapultide,KL4 in lucinactant, Sincalide, Somatorelin acetate, GHRH, GHRF, GRF,Somatostatin acetate, Spaglumat magnesium (or sodium) salt, Substance P,Taltirelin hydrate, Teduglutide, Teicoplanin, Telavancin, Teriparatide,Terlipressin acetate, Tetracosactide, Thymalfasin, thymosin a-1,Thymopentin, Trebananib, Triptorelin, Triptorelin pamoate,Tyroserleutide, Ularitide, Vancomycin, Vapreotide acetate, Vasoactiveintestinal peptide acetate, Vx-001c, TERT572Y, Ziconotide acetate, α5-α6Bax peptide, and β-defensin.

In some embodiments, the peptide is any peptide which would assist inachieving a desired purpose with the composition. For example, in someembodiments, the peptide is any peptide that will facilitate treatmentof any type of disease and/or disorder (e.g., peripheral ischemia,cancer, inflammatory disorders, genetic disorders, etc.).

In some embodiments, the biomacromolecule agent is a nucleic acid. Suchembodiments encompass any type of nucleic acid molecule including, butnot limited to, RNA, siRNA, microRNA, interference RNA, mRNA, repliconmRNA, RNA-analogues, and DNA.

In certain embodiments, the present invention provides methods fortreating conditions, disorders and/or diseases with such compositionscomprising a nanoparticle (as described herein), wherein any kind ofbiomacromolecule is associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) the nanoparticle.

The present invention is not limited to specific types of conditions,disorders and/or diseases.

In some embodiments, the condition, disorder and/or disease isperipheral ischemia, cancer, an inflammatory disorder, a geneticdisorder, etc.

In some embodiments, the condition, disorder and/or disease is selectedfrom erythropoietic porphyries, T2 diabetes, antifibrinolytic, centraldiabetes insipidus, delaying the birth in case of threat of prematurebirth, antibiotic, cystic fibrosis, angina, anticoagulant in patientswith unstable angina undergoing PTCA or PCI, systemic lupuserythematosus, hypercalcemia, osteoporosis, pagets disease, carbetocinworks as an oxytocic, antihemorrhagic and uterotonic drug in theperipheral nervous system, prevention of uterine atony, induction, andcontrol postpartum bleeding or hemorrhage, stimulant of the gastricsecretion, for treat hormone-sensitive cancers of the prostate andbreast, inhibition of premature LH surges in women undergoing controlledovarian stimulation, immunosuppression in organ transplantation toprevent rejection, peritumoral brain edema, diagnosis of ACTHdependentCushing's syndrome, allergies, ankylosing spondylitis, psoriasis,chorioditis, erythema, keratitis, sclerosis, dermatomyositis, rheumatoidarthritis, Stevens-Johnson Syndrome, ulcerative colitis, diagnosis ofadrenocortical insufficiency, antibiotic, systemic infections caused bygram positive organisms, nocturnal enuresis, nocturia, and stoppage ofbleeding or hemorrhage in haemophilia A patients, acute hereditaryangioderma, postmenopausal osteoporosis, anti-parathyroid, Paget'sdisease, hypercalcaemia, hypertension, AIDS/HIV-1 infection, acutecoronary syndrome, unstable angina undergoing PCI, Alzheimer's andParkinson's disease, inhibition of premature LH surges in womenundergoing controlled ovarian hyperstimulation, Relapsing-RemittingMultiple Sclerosis, hepatic insufficiency, wound healing, inflammationof respiratory tract, asthenia, release of follicle-stimulating hormone(FSH) and luteinizing hormone (LH) from the anterior pituitary,stimulate the secretion of gonadotropin during disturbances fertility,and diagnosis of the functional capacity and response of thegonadotropes of the anterior pituitary, for skin lesions, surface woundsand eye infections, postmenopausal osteoporosis, Paget's disease,hypercalcaemia, hereditary angioedema, immune system related diseases,acromegaly, anticoagulant, fibroids and endometriosis, central diabetesinsipidus, Cushing's syndrome, diabetic foot ulcers, treatment ofcentral precocious puberty, uterine fibriods and endometriosis,vasodilatory, natriuretic, diuretic and neurohormonal effects,acromegaly, carcinoid syndrome, acute bacterial skin and skin structureinfections, initiation or improvement of uterine contractions, andcontrol postpartum bleeding or hemorrhage, hematide Chronic kidneydisease associated anemia, stomatitis, pharyngitis, diagnosticassessment of thyroid function, postmenopausal osteoporosis,hypercalcaemia, diagnosis of pancreatic exocrine dysfunction, andgastrinoma, Zollinger-Ellison syndrome, prevention of RDS in prematureinfants, and meconium aspiration syndrome, acute variceal bleeding,allergic rhinitis and conjunctivitis, spinocerebellardegeneration/ataxia, Short Bowel Syndrome, antibiotic, bactericidal,teriparatide is the only anabolic (i.e., bone growing) agent indicatedfor use in postmenopausal women with osteoporosis, Cortrosyn Analogue ofadrenocorticotrophic hormone (ACTH) used for diagnostic purposes,treatment of adrenal insufficiency, epilepsia, Chronic hepatitis B,chronic hepatitis C, primary and secondary immune deficiencies, acutedecompensated heart failure, colitis, esophageal variceal bleeding inpatients with cirrhotic liver disease and AIDS-related diarrhea,sarcoidosis and acute lung injury, and severe chronic pain.

In some embodiments, the following Table 1 recites a peptide anddisorder and/or disease to be treated.

Immune Response Stimulation

The immune system can be classified into two functional subsystems: theinnate and the acquired immune system. The innate immune system is thefirst line of defense against infections, and most potential pathogensare rapidly neutralized by this system before they can cause, forexample, a noticeable infection. The acquired immune system reacts tomolecular structures, referred to as antigens, of the intrudingorganism. There are two types of acquired immune reactions, whichinclude the humoral immune reaction and the cell-mediated immunereaction. In the humoral immune reaction, antibodies secreted by B cellsinto bodily fluids bind to pathogen-derived antigens, leading to theelimination of the pathogen through a variety of mechanisms, e.g.complement-mediated lysis. In the cell-mediated immune reaction, T-cellscapable of destroying other cells are activated. For example, ifproteins associated with a disease are present in a cell, they arefragmented proteolytically to peptides within the cell. Specific cellproteins then attach themselves to the antigen or peptide formed in thismanner and transport them to the surface of the cell, where they arepresented to the molecular defense mechanisms, in particular T-cells, ofthe body. Cytotoxic T cells recognize these antigens and kill the cellsthat harbor the antigens.

The molecules that transport and present peptides on the cell surfaceare referred to as proteins of the major histocompatibility complex(MHC). MHC proteins are classified into two types, referred to as MHCclass I and MHC class II. The structures of the proteins of the two MHCclasses are very similar; however, they have very different functions.Proteins of MHC class I are present on the surface of almost all cellsof the body, including most tumor cells. MHC class I proteins are loadedwith antigens that usually originate from endogenous proteins or frompathogens present inside cells, and are then presented to naive orcytotoxic T-lymphocytes (CTLs). MHC class II proteins are present ondendritic cells, B-lymphocytes, macrophages and other antigen-presentingcells. They mainly present peptides, which are processed from externalantigen sources, i.e. outside of the cells, to T-helper (Th) cells. Mostof the peptides bound by the MHC class I proteins originate fromcytoplasmic proteins produced in the healthy host cells of an organismitself, and do not normally stimulate an immune reaction. Accordingly,cytotoxic T-lymphocytes that recognize such self-peptide-presenting MHCmolecules of class I are deleted in the thymus (central tolerance) or,after their release from the thymus, are deleted or inactivated, i.e.tolerized (peripheral tolerance). MHC molecules are capable ofstimulating an immune reaction when they present peptides tonon-tolerized T-lymphocytes. Cytotoxic T-lymphocytes have both T-cellreceptors (TCR) and CD8 molecules on their surface. T-Cell receptors arecapable of recognizing and binding peptides complexed with the moleculesof MHC class I. Each cytotoxic T-lymphocyte expresses a unique T-cellreceptor which is capable of binding specific MHC/peptide complexes.

The peptide antigens attach themselves to the molecules of MHC class Iby competitive affinity binding within the endoplasmic reticulum, beforethey are presented on the cell surface. Here, the affinity of anindividual peptide antigen is directly linked to its amino acid sequenceand the presence of specific binding motifs in defined positions withinthe amino acid sequence. If the sequence of such a peptide is known, itis possible to manipulate the immune system against diseased cellsusing, for example, peptide vaccines.

Peptide-based cancer vaccines have been extensively investigated due totheir good safety profiles and ease of manufacturing and qualitycontrol. However, their anti-tumor efficacy in clinical trials have beendisappointing, a phenomenon that has been attributed to inefficientcodelivery of Ag peptides and adjuvants to draining lymph nodes (dLNs),and subsequentimmunological tolerance and CTL fratricide (see, e.g.,Toes, R. E., et al., Proc. Natl. Acad. Sci. U. S. A. 93, 7855-7860(1996); Su, M. W., et al., J. Immunol. 151, 658-667 (1993); Melief, C.J. & van der Burg, S. H. Nat. Rev. Cancer 8, 351-360 (2008)). Althoughdepot-forming water-in-oil adjuvant systems can improve immunogenicity(see, e.g., Speiser, D. E. et al. J. Clin. Invest. 115, 739-746 (2005);Fourcade, J. et al. J. Immunother. 31, 781-791 (2008)), boosterimmunizations can cause T-cell sequestration at the vaccine site,causing T-cell exhaustion and deletion (see, e.g., Rezvani, K. et al.Haematologica 96, 432-440 (2011); Hailemichael, Y. et al. Nat. Med. 19,465-472 (2013)). To address these issues, various nanoparticle-basedvaccine systems have been evaluated in animal models (see, e.g., Reddy,S. T. et al. Nat. Biotechnol. 25, 1159-1164 (2007); Li, A. V. et al.Sci. Transl. Med. 5, 204ra130 (2013); Jeanbart, L. et al. Cancer.Immunol. Res. 2, 436-447 (2014); Xu, Z., et al., ACS Nano 8, 3636-3645(2014); Liu, H. et al. Nature 507, 519-522 (2014); Fan, Y. & Moon, J. J.Vaccines (Basel) 3, 662-685 (2015)). However, potential safety concernsand scale-up manufacturing of nanoparticles, especially in a mannersuitable for personalized therapeutics with patient-specificneo-antigens, remain as the major challenges.

Experiments conducted during the course of developing embodiments forthe present invention developed an alternative, simple strategy wherepreformed nanoparticles, with an established clinical manufacturingprocedure and excellent safety profiles in humans, were mixed with Agpeptides and adjuvants to produce “personalized” cancer vaccines (FIG.12). The strategy was based on synthetic high density lipoprotein (sHDL)nanodiscs, composed of phospholipids and apolipoprotein A1(ApoA1)-mimetic peptides. Compared with other HDLs containing 243-aminoacid ApoA1 purified from human plasma or produced recombinantly (see,e.g., Wolfrum, C. et al. Nat. Biotechnol. 25, 1149-1157 (2007);Diditchenko, S. et al. Arterioscler. Thromb. Vasc. Biol. 33, 2202-2211(2013); Fischer, N. O. et al. J. Am. Chem. Soc. 135, 2044-2047 (2013);Tardy, C. et al. Atherosclerosis 232, 110-118 (2014); Duivenvoorden, R.et al. Nat. Commun. 5, 3065 (2014)), sHDL nanodiscs were synthesizedwith 22-mer peptides (22A), derived from the repeat α-helix domain ofApoA1 (see, e.g,. U.S. Pat. Nos. 6,734,169; 8,378,068; Li, D., Gordon,S., Schwendeman, A. & Remaley, A. T. Apolipoprotein mimetic peptides forstimulating cholesterol efflux. in Apolipoprotein Mimetics in theManagement of Human Disease (eds. Anantharamaiah, G. M. & Goldberg, D.)29-42 (Springer, Switzerland, 2015)), with no sequence homology toendogenous ApoA1, thus averting potential trigger of autoimmunity.Importantly, sHDL has been previously manufactured for clinical testingand proven to be safe in humans with the maximum tolerated dose at ˜2.2g/m² (see, e.g., Khan, M., et al., Circulation 108 (Suppl IV), 563-564(2003); Miles, J., et al. Proceedings of Arteriosclerosis Thrombosis andVascular Biology 24, E19-E19 (2004), a value one- to two-orders ofmagnitude greater than most polymeric or inorganic nanoparticles inclinical trials (see, e.g., Alexis, F., et al., Mol. Pharm. 5, 505-515(2008); Anselmo, A. C. & Mitragotri, S. A, AAPS J 17, 1041-1054 (2015).

Experiments conducted during the course of developing embodiments forthe present invention developed a nanodisc-based platform forneo-antigen vaccination (FIG. 12). Exploiting the endogenous role of HDLas a nanoparticle for cholesterol, immunostimulatory agent CpG, a strongToll-like receptor-9 agonist, was modified with cholesterol (Cho-CpG) toenhance its in vivo trafficking. It was shown that preformed sHDLnanodiscs can be simply mixed with cholesteryl-CpG and tumor Agpeptides, including neo-antigens identified via tumor DNA sequencing, toproduce homogeneous, stable, and ultrasmall nanodiscs in <2 h at roomtemperature (RT). The nanodiscs efficiently promoted co-delivery ofAg/CpG to dLNs, prolonged Ag presentation on antigen-presenting cells(APCs), and elicited striking levels of CTL responses with anti-tumorefficacy. Owning to the facile production process, robust therapeuticefficacy, and clinical safety demonstrated previously (see, e.g., Khan,M., et al., Circulation 108 (Suppl IV), 563-564 (2003); Miles, J., etal. Proceedings of Arteriosclerosis Thrombosis and Vascular Biology 24,E19-E19 (2004)), this approach offers an attractive platform technologyfor patient-tailored cancer vaccines as well as other bioactivetherapeutics.

Accordingly, in certain embodiments, nanoparticles (e.g., sHDLnanoparticles) associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) an antigen are used forinducing an immune response. In some embodiments, the nanoparticles arefurther associated with (e.g., complexed, conjugated, encapsulated,absorbed, adsorbed, admixed) an adjuvant (e.g., dendritic cell targetingmolecule (DC)). In some embodiments, the nanoparticles areco-administered with an adjuvant. In some embodiments, the antigen isassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the adjuvant. In some embodiments, the antigen is notassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) the adjuvant. In some embodiments, the antigen isconjugated with a hydrophobic molecule. In some embodiments, theadjuvant is conjugated with a hydrophobic molecule. In some embodiments,the average size of the nanoparticle is between 6 to 500 nm.

In some embodiments, the hydrophobic molecule is a lipid molecule. Insome embodiments, the lipid molecule is a membrane-forming lipidmolecule. In some embodiments, the lipid molecule molecule is anon-membrane-forming lipid molecule.

Examples of lipid molecules applicable with the embodiments of thepresent invention include, but are not limited to, phospholipids such aslecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Other non-limiting examples of lipid molecules include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.

Other examples of lipid molecules suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

Other examples of lipid molecules suitable for use in the presentinvention include fatty acids and derivatives or analogs thereof. Theyinclude oleic acid, lauric acid, capric acid (n-decanoic acid), myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,caprylic acid, arachidonic acid, glycerol 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkylesters thereof (e.g., methyl, isopropyl and t-butyl), and mono- anddi-glycerides thereof (i.e., oleate, laurate, caprate, myristate,palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri etal., J. Pharm. Pharmacol., 1992, 44, 651-654).

Other examples of lipid molecules suitable for use in the presentinvention include a lipid molecule modified with PEG (PEG-lipid).Examples of PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes. AdditionalPEG-lipids include, without limitation, PEG-C-DOMG, 2 KPEG-DMG, and amixture thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). OtherPEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150(e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipidconjugates of the present invention. The disclosures of these patentsare herein incorporated by reference in their entirety for all purposes.In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CH₂COOH) isparticularly useful for preparing PEG-lipid conjugates including, e.g.,PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.

Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

Such embodiments are not limited to particular antigen. Indeed, antigenscan be peptides, proteins, polysaccharides, saccharides, lipids,glycolipids, nucleic acids, or combinations thereof. The antigen can bederived from any source, including, but not limited to, a virus,bacterium, parasite, plant, protozoan, fungus, tissue or transformedcell such as a cancer or leukemic cell and can be a whole cell orimmunogenic component thereof, e.g., cell wall components or molecularcomponents thereof.

In some embodiments, the antigens are known in the art and are availablefrom commercial government and scientific sources. In some embodiments,the antigens are whole inactivated or attenuated organisms. Theseorganisms may be infectious organisms, such as viruses, parasites andbacteria. These organisms may also be tumor cells. The antigens may bepurified or partially purified polypeptides derived from tumors or viralor bacterial sources. Criteria for identifying and selecting effectiveantigenic peptides (e.g., minimal peptide sequences capable of elicitingan immune response) can be found in the art. The antigens can berecombinant polypeptides produced by expressing DNA encoding thepolypeptide antigen in a heterologous expression system. The antigenscan be DNA encoding all or part of an antigenic protein. The DNA may bein the form of vector DNA such as plasmid DNA.

Antigens may be provided as single antigens or may be provided incombination. Antigens may also be provided as complex mixtures ofpolypeptides or nucleic acids.

In some embodiments, the antigen is a self antigen. As used herein, theterm “self-antigen” refers to an immunogenic antigen or epitope which isnative to a mammal and which may be involved in the pathogenesis of anautoimmune disease.

In some embodiments, the antigen is a viral antigen. Viral antigens canbe isolated from any virus including, but not limited to, a virus fromany of the following viral families: Arenaviridae, Arterivirus,Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae,Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus,Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae(e.g., Coronavirus, such as severe acute respiratory syndrome (SARS)virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus,Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g.,Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g.,Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, andDengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae,Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g.,Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g.,measles, mumps, and human respiratory syncytial virus), Parvoviridae,Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, andaphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae(e.g., rotavirus), Retroviridae (e.g., lentivirus, such as humanimmunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example,rabies virus, measles virus, respiratory syncytial virus, etc.),Togaviridae (for example, rubella virus, dengue virus, etc.), andTotiviridae. Suitable viral antigens also include all or part of Dengueprotein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and DengueD1NS3.

Viral antigens may be derived from a particular strain such as apapilloma virus, a herpes virus, i.e. herpes simplex 1 and 2; ahepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus(HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV),hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borneencephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus,Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, and lymphocytic choriomeningitis.

In some embodiments, the antigen is a bacterial antigen. Bacterialantigens can originate from any bacteria including, but not limited to,Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella,Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium,Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia,Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilusinfluenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis,Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus,Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter,Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum,Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta,Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma,Thiobacillus, and Treponema, Vibrio, and Yersinia.

In some embodiments, the antigen is a parasite antigen. Parasiteantigens can be obtained from parasites such as, but not limited to, anantigen derived from Cryptococcus neoformans, Histoplasma capsulatum,Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsiaricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci,Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei,Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis andSchistosoma mansoni. These include Sporozoan antigens, Plasmodianantigens, such as all or part of a Circumsporozoite protein, aSporozoite surface protein, a liver stage antigen, an apical membraneassociated protein, or a Merozoite surface protein.

In some embodiments, the antigen is an allergen and environmentalantigen, such as, but not limited to, an antigen derived from naturallyoccurring allergens such as pollen allergens (tree-, herb, weed-, andgrass pollen allergens), insect allergens (inhalant, saliva and venomallergens), animal hair and dandruff allergens, and food allergens.Important pollen allergens from trees, grasses and herbs originate fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding i.a. birch (Betula), alder (Alnus), hazel (Corylus), hornbeam(Carpinus) and olive (Olea), cedar (Cryptomeria and Jumperus), Planetree (Platanus), the order of Poales including i.e. grasses of thegenera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale,and Sorghum, the orders of Asterales and Urticales including i. a. herbsof the genera Ambrosia, Artemisia, and Parietaria. Other allergenantigens that may be used include allergens from house dust mites of thegenus Dermatophagoides and Euroglyphus, storage mite e.g Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, those frommammals such as cat, dog and horse, birds, venom allergens includingsuch originating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (superfamily Apidae),wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Stillother allergen antigens that may be used include inhalation allergensfrom fungi such as from the genera Alternaria and Cladosporium.

In some embodiments, the antigen is a tumor antigen. The antigen can bea tumor antigen, including a tumor-associated or tumor-specific antigen,such as, but not limited to, alpha-actinin-4, Bcr-Abl fusion protein,Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusionprotein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusionprotein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3,neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras,N-ras, Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7, GnTV,Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88,NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1,GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGS), SCP-1, Hom/Mel-40, PRAME,p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR,Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigensE6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3,c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F,5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,G250, Ga733 (EpCAM), human EGFR protein or its fragments, such as humanEGFR residues 306-325 (SCVRACGADSYEMEEDGVRK (SEQ ID NO:374)) andresidues 897-915 (VWSYGVTVWELMTFGSKPY (SEQ ID NO:375)), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,TPS, WT1 (and WT1-derived peptide sequences: WT1 126-134 (RMFP NAPYL(SEQ ID NO:376)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO:377)), andWT1 122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO:378)), MUC1 (andMUC1-derived peptides and glycopeptides such as RPAPGS (SEQ ID NO:379),PPAHGVT (SEQ ID NO:380), and PDTRP (SEQ ID NO:381))), LMP2, EGFRvIII,Idiotype, GD2, Ras mutant, p53 mutant, Proteinase3 (PR1), Survivin,hTERT, Sarcoma translocation breakpoints, EphA2, EphA4, LMW-PTP, PAP,ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgenreceptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, FucosylGM1, Mesothelin, sLe(animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH,NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Spermprotein 17, LCK, HMWMAA, AKAP-4, XAGE 1, B7H3, Legumain, Tie 2, Page4,VEGFR2, MAD-CT-1, FAP, PDGFR-alpha, PDGFR-β, MAD-CT-2, Fos-relatedantigen 1, ERBB2, Folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K,fms-related tyro-sine kinase 1 (FLT1, best known as VEGFR1), KDR, PADRE,TA-CIN (recombinant HPV16 L2E7E6), SOX2, and aldehyde dehydrogenase.

One of the critical barriers to developing curative and tumor-specificimmunotherapy is the identification and selection of highly specific andrestricted tumor antigens to avoid autoimmunity. Tumor neo-antigens,which arise as a result of genetic change (e.g., inversions,translocations, deletions, missense mutations, splice site mutations,etc.) within malignant cells, represent the most tumor-specific class ofantigens.

In some embodiments, the antigen is a neo-antigen. The term neoantigenis used herein to define any newly expressed antigenic determinant.Neoantigens may arise upon conformational change in a protein, as newlyexpressed determinants (especially on the surfaces of transformed orinfected cells), as the result of complex formation of one or moremolecules or as the result of cleavage of a molecule with a resultantdisplay of new antigenic determinants. Thus, as used herein, the termneoantigen covers antigens expressed upon infection (e.g. viralinfection, protozoal infection or bacterial infection), inprion-mediated diseases, an on cell transformation (cancer), in whichlatter case the neoantigen may be termed a tumor-associated antigen.

The present invention is not limited to a particular manner ofidentifying neo-antigens. In some embodiments, identification ofneo-antigens involves identifying all, or nearly all, mutations in theneoplasia/tumor at the DNA level using whole genome sequencing, wholeexome (e.g., only captured exons) sequencing, or RNA sequencing of tumorversus matched germline samples from each patient. In some embodiments,identification of neo-antigens involves analyzing the identifiedmutations with one or more peptide-MHC binding prediction algorithms togenerate a plurality of candidate neo-antigen T cell epitopes that areexpressed within the neoplasia/tumor and may bind patient HLA alleles.In some embodiments, identification of neo-antigens involvessynthesizing the plurality of candidate neo-antigen peptides selectedfrom the sets of all neo open reading frame peptides and predictedbinding peptides for use in a cancer vaccine.

As such, the present invention is based, at least in part, on theability to identify all, or nearly all, of the mutations within aneoplasia/tumor (e.g., translocations, inversions, large and smalldeletions and insertions, missense mutations, splice site mutations,etc.). In particular, these mutations are present in the genome ofneoplasia/tumor cells of a subject, but not in normal tissue from thesubject. Such mutations are of particular interest if they lead tochanges that result in a protein with an altered amino acid sequencethat is unique to the patient's neoplasia/tumor (e.g., a neo-antigen).For example, useful mutations may include: (1) non-synonymous mutationsleading to different amino acids in the protein; (2) read-throughmutations in which a stop codon is modified or deleted, leading totranslation of a longer protein with a novel tumor-specific sequence atthe C-terminus; (3) splice site mutations that lead to the inclusion ofan intron in the mature mRNA and thus a unique tumor-specific proteinsequence; (4) chromosomal rearrangements that give rise to a chimericprotein with tumor-specific sequences at the junction of 2 proteins(i.e., gene fusion); (5) frameshift mutations or deletions that lead toa new open reading frame with a novel tumor-specific protein sequence;and the like. Peptides with mutations or mutated polypeptides arisingfrom, for example, splice-site, frameshift, read-through, or gene fusionmutations in tumor cells may be identified by sequencing DNA, RNA orprotein in tumor versus normal cells.

Also within the scope of the present invention is personal neo-antigenpeptides derived from common tumor driver genes and may further includepreviously identified tumor specific mutations.

Preferably, any suitable sequencing-by-synthesis platform can be used toidentify mutations. Four major sequencing-by-synthesis platforms arecurrently available: the Genome Sequencers from Roche/454 Life Sciences,the HiSeq Analyzer from Illumina/Solexa, the SOLiD system from AppliedBioSystems, and the Heliscope system from Helicos Biosciences.Sequencing-by-synthesis platforms have also been described by PacificBiosciences and VisiGen Biotechnologies. Each of these platforms can beused in the methods of the invention. In some embodiments, a pluralityof nucleic acid molecules being sequenced is bound to a support (e.g.,solid support). To immobilize the nucleic acid on a support, a capturesequence/universal priming site can be added at the 3′ and/or 5′ end ofthe template. The nucleic acids may be bound to the support byhybridizing the capture sequence to a complementary sequence covalentlyattached to the support. The capture sequence (also referred to as auniversal capture sequence) is a nucleic acid sequence complementary toa sequence attached to a support that may dually serve as a universalprimer.

As an alternative to a capture sequence, a member of a coupling pair(such as, e.g., antibody/antigen, receptor/ligand, or the avidin-biotinpair as described in, e.g., U.S. Patent Application No. 2006/0252077)may be linked to each fragment to be captured on a surface coated with arespective second member of that coupling pair. Subsequent to thecapture, the sequence may be analyzed, for example, by single moleculedetection/sequencing, e.g., as described in the Examples and in U.S.Pat. No. 7,283,337, including template-dependentsequencing-by-synthesis. In sequencing-by-synthesis, the surface-boundmolecule is exposed to a plurality of labeled nucleotide triphosphatesin the presence of polymerase. The sequence of the template isdetermined by the order of labeled nucleotides incorporated into the 3′end of the growing chain. This can be done in real time or in astep-and-repeat mode. For real-time analysis, different optical labelsto each nucleotide may be incorporated and multiple lasers may beutilized for stimulation of incorporated nucleotides.

Any cell type or tissue may be utilized to obtain nucleic acid samplesfor use in the sequencing methods described herein. In some embodiments,the DNA or RNA sample is obtained from a neoplasia/tumor or a bodilyfluid, e.g., blood, obtained by known techniques (e.g. venipuncture) orsaliva. Alternatively, nucleic acid tests can be performed on drysamples (e.g. hair or skin).

A variety of methods are available for detecting the presence of aparticular mutation or allele in an individual's DNA or RNA.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

PCR based detection means may include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously.

Alternatively, it is possible to amplify different markers with primersthat are differentially labeled and thus can each be differentiallydetected. Of course, hybridization based detection means allow thedifferential detection of multiple PCR products in a sample. Othertechniques are known in the art to allow multiplex analyses of aplurality of markers.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms in genomic DNA or cellular RNA. In oneembodiment, the single base polymorphism can be detected by using aspecialized exonuclease-resistant nucleotide, as disclosed, e.g., U.S.Pat. No. 4,656,127. According to the method, a primer complementary tothe allelic sequence immediately 3′ to the polymorphic site is permittedto hybridize to a target molecule obtained from a particular animal orhuman. If the polymorphic site on the target molecule contains anucleotide that is complementary to the particular exonuclease-resistantnucleotide derivative present, then that derivative will be incorporatedonto the end of the hybridized primer. Such incorporation renders theprimer resistant to exonuclease, and thereby permits its detection.Since the identity of the exonuclease-resistant derivative of the sampleis known, a finding that the primer has become resistant to exonucleasesreveals that the nucleotide present in the polymorphic site of thetarget molecule was complementary to that of the nucleotide derivativeused in the reaction. This method has the advantage that it does notrequire the determination of large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site(see, e.g, French Patent No. 2,650,840; PCT Application No.WO1991/02087). As in the method of U.S. Pat. No. 4,656,127, a primer maybe employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site, will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA® isdescribed in PCT Application No. WO 1992/15712). GBA® uses mixtures oflabeled terminators and a primer that is complementary to the sequence3′ to a polymorphic site. The labeled terminator that is incorporated isthus determined by, and complementary to, the nucleotide present in thepolymorphic site of the target molecule being evaluated. In contrast tothe method of Cohen et al. (French Patent 2,650,840; PCT Application No.W01991/02087) the GBA® method is preferably a heterogeneous phase assay,in which the primer or the target molecule is immobilized to a solidphase. Recently, several primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DN A have been described(see, e.g., Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989);Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C, et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88: 1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat.1: 159-164 (1992); Ugozzoli, L. et al., GATA 9: 107-112 (1992); Nyren,P. et al., Anal. Biochem. 208: 171-175 (1993)). These methods differfrom GBA® in that they all rely on the incorporation of labeleddeoxynucleotides to discriminate between bases at a polymorphic site. Insuch a format, since the signal is proportional to the number ofdeoxynucleotides incorporated, polymorphisms that occur in runs of thesame nucleotide can result in signals that are proportional to thelength of the run (see, e.g., Syvanen, A.-C, et al., Amer. J. Hum.Genet. 52:46-59 (1993)).

An alternative method for identifying tumor specific neo-antigens isdirect protein sequencing. Protein sequencing of enzymatic digests usingmultidimensional MS techniques (MSn) including tandem mass spectrometry(MS/MS)) can also be used to identify neo-antigens of the invention.Such proteomic approaches permit rapid, highly automated analysis (see,e.g., K. Gevaert and J. Vandekerckhove, Electrophoresis 21: 1145-1154(2000)). It is further contemplated within the scope of the inventionthat high-throughput methods for de novo sequencing of unknown proteinsmay be used to analyze the proteome of a patient's tumor to identifyexpressed neo-antigens. For example, meta shotgun protein sequencing maybe used to identify expressed neo-antigens (see, e.g., Guthals et al.(2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecularand Cellular Proteomics 11(10): 1084-96).

Tumor specific neo-antigens may also be identified using MHC multimersto identify neo-antigen-specific T-cell responses. For example,highthroughput analysis of neo-antigen-specific T-cell responses inpatient samples may be performed using MHC tetramer-based screeningtechniques (see, e.g., Hombrink et al. (2011) High-ThroughputIdentification of Potential Minor Histocompatibility Antigens by MHCTetramer-Based Screening: Feasibility and Limitations 6(8): 1-11; Hadrupet al. (2009) Parallel detection of antigen-specific T-cell responses bymultidimensional encoding of MHC multimers, Nature Methods, 6(7):520-26;van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specificT-cell reactivity in an Ipilimumab-responsive melanoma, Journal ofClinical Oncology, 31: 1-4; and Heemskerk et al. (2013) The cancerantigenome, EMBO Journal, 32(2): 194-203). It is contemplated within thescope of the invention that such tetramer-based screening techniques maybe used for the initial identification of tumor specific neo-antigens,or alternatively as a secondary screening protocol to assess whatneo-antigens a patient may have already been exposed to, therebyfacilitating the selection of candidate neo-antigens for the vaccines ofthe invention.

The invention further includes isolated peptides (e.g., neo-antigenicpeptides containing the tumor specific mutations identified by thedescribed methods, peptides that comprise known tumor specificmutations, and mutant polypeptides or fragments thereof identified bythe described methods). These peptides and polypeptides are referred toherein as “neo-antigenic peptides” or “neo-antigenic polypeptides.” Thepolypeptides or peptides can be of a variety of lengths and willminimally include the small region predicted to bind to the HLA moleculeof the patient (the “epitope”) as well as additional adjacent aminoacids extending in both the N- and C-terminal directions. Thepolypeptides or peptides can be either in their neutral (uncharged)forms or in forms which are salts, and either free of modifications suchas glycosylation, side chain oxidation, or phosphorylation or containingthese modifications, subject to the condition that the modification notdestroy the biological activity of the polypeptides as herein described.

In certain embodiments the size of the at least one neo-antigenicpeptide molecule may comprise, but is not limited to, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 60,about 70, about 80, about 90, about 100, about 110, about 120 or greateramino molecule residues, and any range derivable therein. In specificembodiments the neo-antigenic peptide molecules are equal to or lessthan 50 amino acids. In a preferred embodiment, the neo-antigenicpeptide molecules are equal to about 20 to about 30 amino acids.

As such, the present invention provides nanoparticles (e.g., sHDLnanoparticles) associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) one or more neo-antigenicpeptides. In some embodiments, the nanoparticle (e.g., sHDLnanoparticle) is associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) one neo-antigenic peptide. Insome embodiments, the nanoparticle (e.g., sHDL nanoparticle) isassociated with (e.g., complexed, conjugated, encapsulated, absorbed,adsorbed, admixed) two neo-antigenic peptides. In some embodiments, thenanoparticle (e.g., sHDL nanoparticle) is associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) atleast 5 or more neo-antigenic peptides. In some embodiments, thenanoparticle (e.g., sHDL nanoparticle) is associated with (e.g.,complexed, conjugated, encapsulated, absorbed, adsorbed, admixed) atleast about 6, about 8, about 10, about 12, about 14, about 16, about18, or about 20 distinct peptides. In some embodiments, the nanoparticle(e.g., sHDL nanoparticle) is associated with (e.g., complexed,conjugated, encapsulated, absorbed, adsorbed, admixed) at least 20distinct peptides.

The neo-antigenic peptides, polypeptides, and analogs can be furthermodified to contain additional chemical moieties not normally part ofthe protein. Those derivatized moieties can improve the solubility, thebiological half-life, absorption of the protein, or binding affinity.The moieties can also reduce or eliminate any desirable side effects ofthe proteins and the like. An overview for those moieties can be foundin Remington's Pharmaceutical Sciences, 20^(th) ed., Mack PublishingCo., Easton, Pa. (2000). For example, neo-antigenic peptides andpolypeptides having the desired activity may be modified as necessary toprovide certain desired attributes, e.g. improved pharmacologicalcharacteristics, while increasing or at least retaining substantiallyall of the biological activity of the unmodified peptide to bind thedesired MHC molecule and activate the appropriate T cell. For instance,the neo-antigenic peptide and polypeptides may be subject to variouschanges, such as substitutions, either conservative or non-conservative,where such changes might provide for certain advantages in their use,such as improved MHC binding. Such conservative substitutions mayencompass replacing an amino acid residue with another amino acidresidue that is biologically and/or chemically similar, e.g., onehydrophobic residue for another, or one polar residue for another. Theeffect of single amino acid substitutions may also be probed usingD-amino acids. Such modifications may be made using well known peptidesynthesis procedures, as described in e.g., Merrifield, Science232:341-347 (1986), Barany & Merrifield, The Peptides, Gross &Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart &Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed.(1984).

In some embodiments, the neo-antigenic peptides and polypeptides may bemodified with linking agents for purposes of facilitating associationwith the nanoparticle (e.g., sHDL nanoparticle). The invention is notlimited to a particular type or kind of linking agent. In someembodiments, the linking agent is a cysteine-serine-serine (CSS)molecule.

In some embodiments wherein the nanoparticle is sHDL and theneo-antigenic peptide or polypeptide is modified with CSS, the sHDL isfurther modified withdioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP) wherein upon mixing, the DOPE-PDP and CSS engagethereby resulting in a complexing (linking) of the CSS-Ag with the sHDL.

The neo-antigenic peptide and polypeptides may also be modified byextending or decreasing the compound's amino acid sequence, e.g., by theaddition or deletion of amino acids. The neo-antigenic peptides,polypeptides, or analogs can also be modified by altering the order orcomposition of certain residues. It will be appreciated by the skilledartisan that certain amino acid residues essential for biologicalactivity, e.g., those at critical contact sites or conserved residues,may generally not be altered without an adverse effect on biologicalactivity. The non-critical amino acids need not be limited to thosenaturally occurring in proteins, such as L-a-amino acids, or theirD-isomers, but may include non-natural amino acids as well, such asβ-γ-δ-amino acids, as well as many derivatives of L-a-amino acids.

Typically, a neo-antigen polypeptide or peptide may be optimized byusing a series of peptides with single amino acid substitutions todetermine the effect of electrostatic charge, hydrophobicity, etc. onMHC binding. For instance, a series of positively charged (e.g., Lys orArg) or negatively charged (e.g., Glu) amino acid substitutions may bemade along the length of the peptide revealing different patterns ofsensitivity towards various MHC molecules and T cell receptors. Inaddition, multiple substitutions using small, relatively neutralmoieties such as Ala, Gly, Pro, or similar residues may be employed. Thesubstitutions may be homo-oligomers or hetero-oligomers. The number andtypes of residues which are substituted or added depend on the spacingnecessary between essential contact points and certain functionalattributes which are sought (e.g., hydrophobicity versushydrophilicity). Increased binding affinity for an MHC molecule or Tcell receptor may also be achieved by such substitutions, compared tothe affinity of the parent peptide. In any event, such substitutionsshould employ amino acid residues or other molecular fragments chosen toavoid, for example, steric and charge interference which might disruptbinding. Amino acid substitutions are typically of single residues.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final peptide.

One of skill in the art will appreciate that there are a variety of waysin which to produce such tumor specific neo-antigens. In general, suchtumor specific neo-antigens may be produced either in vitro or in vivo.Tumor specific neo-antigens may be produced in vitro as peptides orpolypeptides, which may then be formulated into a personalized neoplasiavaccine and administered to a subject. Such in vitro production mayoccur by a variety of methods known to one of skill in the art such as,for example, peptide synthesis or expression of a peptide/polypeptidefrom a DNA or RNA molecule in any of a variety of bacterial, eukaryotic,or viral recombinant expression systems, followed by purification of theexpressed peptide/polypeptide.

Alternatively, tumor specific neo-antigens may be produced in vivo byintroducing molecules (e.g., DNA, RNA, viral expression systems, and thelike) that encode tumor specific neo-antigens into a subject, whereuponthe encoded tumor specific neo-antigens are expressed.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases located at theNational Institutes of Health website. The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

Peptides can be readily synthesized chemically utilizing reagents thatare free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Am. Chem. Soc. 85:2149-54, 1963).

A further aspect of the invention provides a nucleic acid (e.g., apolynucleotide) encoding a neo-antigenic peptide of the invention, whichmay be used to produce the neo-antigenic peptide in vitro. Thepolynucleotide may be, e.g., DNA, cDNA, PNA, CNA, RNA, either single-and/or double-stranded, or native or stabilized forms ofpolynucleotides, such as e.g. polynucleotides with a phosphorothiatebackbone, or combinations thereof and it may or may not contain intronsso long as it codes for the peptide. A still further aspect of theinvention provides an expression vector capable of expressing apolypeptide according to the invention. Expression vectors for differentcell types are well known in the art and can be selected without undueexperimentation. Generally, the DNA is inserted into an expressionvector, such as a plasmid, in proper orientation and correct readingframe for expression. If necessary, the DNA may be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host (e.g., bacteria),although such controls are generally available in the expression vector.The vector is then introduced into the host bacteria for cloning usingstandard techniques (see, e.g., Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

The invention further embraces variants and equivalents which aresubstantially homologous to the identified tumor specific neo-antigensdescribed herein. These can contain, for example, conservativesubstitution mutations, i.e., the substitution of one or more aminoacids by similar amino acids. For example, conservative substitutionrefers to the substitution of an amino acid with another within the samegeneral class such as, for example, one acidic amino acid with anotheracidic amino acid, one basic amino acid with another basic amino acid,or one neutral amino acid by another neutral amino acid. What isintended by a conservative amino acid substitution is well known in theart.

The invention also includes expression vectors comprising the isolatedpolynucleotides, as well as host cells containing the expressionvectors. It is also contemplated within the scope of the invention thatthe neo-antigenic peptides may be provided in the form of RNA or cDNAmolecules encoding the desired neo-antigenic peptides. The inventionalso provides that the one or more neo-antigenic peptides of theinvention may be encoded by a single expression vector. The inventionalso provides that the one or more neo-antigenic peptides of theinvention may be encoded and expressed in vivo using a viral basedsystem (e.g., an adenovirus system).

The term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences. The polynucleotides of the invention can be in theform of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, andsynthetic DNA; and can be double-stranded or single-stranded, and ifsingle stranded can be the coding strand or non-coding (anti-sense)strand.

In embodiments, the polynucleotides may comprise the coding sequence forthe tumor specific neo-antigenic peptide fused in the same reading frameto a polynucleotide which aids, for example, in expression and/orsecretion of a polypeptide from a host cell (e.g., a leader sequencewhich functions as a secretory sequence for controlling transport of apolypeptide from the cell). The polypeptide having a leader sequence isa preprotein and can have the leader sequence cleaved by the host cellto form the mature form of the polypeptide.

In some embodiments, the polynucleotides can comprise the codingsequence for the tumor specific neo-antigenic peptide fused in the samereading frame to a marker sequence that allows, for example, forpurification of the encoded polypeptide, which may then be incorporatedinto the personalized neoplasia vaccine. For example, the markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or the marker sequence can be ahemagglutinin (HA) tag derived from the influenza hemagglutinin proteinwhen a mammalian host (e.g., COS-7 cells) is used. Additional tagsinclude, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, Stags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag,SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags,fluorescent protein tags (e.g., green fluorescent protein tags), maltosebinding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Tytag, and the like. In embodiments, the polynucleotides may comprise thecoding sequence for one or more of the tumor specific neo-antigenicpeptides fused in the same reading frame to create a singleconcatamerized neo-antigenic peptide construct capable of producingmultiple neo-antigenic peptides.

In embodiments, the present invention provides isolated nucleic acidmolecules having a nucleotide sequence at least 60% identical, at least65% identical, at least 70% identical, at least 75% identical, at least80% identical, at least 85% identical, at least 90% identical, at least95% identical, or at least 96%, 97%, 98% or 99% identical to apolynucleotide encoding a tumor specific neo-antigenic peptide of thepresent invention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80% identical, at least 85% identical, at least 90% identical,and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

The isolated tumor specific neo-antigenic peptides described herein canbe produced in vitro (e.g., in the laboratory) by any suitable methodknown in the art. Such methods range from direct protein syntheticmethods to constructing a DNA sequence encoding isolated polypeptidesequences and expressing those sequences in a suitable transformed host.In some embodiments, a DNA sequence is constructed using recombinanttechnology by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In embodiments, a DNA sequence encoding a polypeptide of interest wouldbe constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (e.g., by synthesis, site-directed mutagenesis, oranother method), the polynucleotide sequences encoding a particularisolated polypeptide of interest will be inserted into an expressionvector and optionally operatively linked to an expression controlsequence appropriate for expression of the protein in a desired host.Proper assembly can be confirmed by nucleotide sequencing, restrictionmapping, and expression of a biologically active polypeptide in asuitable host. As well known in the art, in order to obtain highexpression levels of a transfected gene in a host, the gene can beoperatively linked to transcriptional and translational expressioncontrol sequences that are functional in the chosen expression host.Recombinant expression vectors may be used to amplify and express DNAencoding the tumor specific neo-antigenic peptides. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a tumor specific neo-antigenicpeptide or a bioequivalent analog operatively linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transforaiants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous, and in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art (see Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23: 175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography, and the like),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence,glutathione-S-transferase, and the like can be attached to the proteinto allow easy purification by passage over an appropriate affinitycolumn. Isolated proteins can also be physically characterized usingsuch techniques as proteolysis, nuclear magnetic resonance and x-raycrystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein. Recombinant protein produced in bacterial culture can beisolated, for example, by initial extraction from cell pellets, followedby one or more concentration, salting-out, aqueous ion exchange or sizeexclusion chromatography steps. High performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of a recombinant protein can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

As such, in certain embodiments, the present invention relates topersonalized strategies for the treatment of disorders (e.g.,neoplasia), and more particularly tumors, by administering atherapeutically effective amount of a sHDL molecule associated with(e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, admixed)one or more neoplasia/tumor specific neo-antigens to a subject (e.g., amammal such as a human) (e.g., a vaccine composition capable of raisinga specific T-cell response). Indeed, in certain embodiments, wholegenome/exome sequencing may be used to identify all, or nearly all,mutated neo-antigens that are uniquely present in a neoplasia/tumor ofan individual patient, and that this collection of mutated neo-antigensmay be analyzed to identify a specific, optimized subset of neo-antigensfor use as a personalized cancer vaccine for treatment of the patient'sneoplasia/tumor. For example, in some embodiments, a population ofneoplasia/tumor specific neo-antigens may be identified by sequencingthe neoplasia/tumor and normal DNA of each patient to identifytumor-specific mutations, and determining the patient's HLA allotype.The population of neoplasia/tumor specific neo-antigens and theircognate native antigens may then be subject to bioinformatic analysisusing validated algorithms to predict which tumor-specific mutationscreate epitopes that could bind to the patient's HLA allotype, and inparticular which tumor-specific mutations create epitopes that couldbind to the patient' s HLA allotype more effectively than the cognatenative antigen. Based on this analysis, one or more peptidescorresponding to a subset of these mutations may be designed andsynthesized for each patient, and pooled together for use as a cancervaccine in immunizing the patient. The neo-antigens peptides may becombined another anti-neoplastic agent. In some embodiments, suchneo-antigens are expected to bypass central thymic tolerance (thusallowing stronger antitumor T cell response), while reducing thepotential for autoimmunity (e.g., by avoiding targeting of normalself-antigens).

The invention further provides a method of inducing a neoplasia/tumorspecific immune response in a subject, vaccinating against aneoplasia/tumor, treating and or alleviating a symptom of cancer in asubject by administering the subject a neo-antigenic peptide or vaccinecomposition of the invention.

According to the invention, the above-described cancer vaccine may beused for a patient that has been diagnosed as having cancer, or at riskof developing cancer. In one embodiment, the patient may have a solidtumor such as breast, ovarian, prostate, lung, kidney, gastric, colon,testicular, head and neck, pancreas, brain, melanoma, and other tumorsof tissue organs and hematological tumors, such as lymphomas andleukemias, including acute myelogenous leukemia, chronic myelogenousleukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, andB cell lymphomas.

The peptide or composition of the invention is administered in an amountsufficient to induce a CTL response. The neo-antigenic peptide,polypeptide or vaccine composition of the invention can be administeredalone or in combination with other therapeutic agents. The therapeuticagent is for example, a chemotherapeutic or biotherapeutic agent,radiation, or immunotherapy. Any suitable therapeutic treatment for aparticular cancer may be administered. Examples of chemotherapeutic andbiotherapeutic agents include, but are not limited to, aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide,filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron,hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan,lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate,metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole,ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine,rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab,vinblastine, vincristine and vinorelbine tartrate. For prostate cancertreatment, a preferred chemotherapeutic agent with which anti-CTLA-4 canbe combined is paclitaxel (Taxol®).

In addition, the subject may be further administered ananti-immunosuppressive or immuno stimulatory agent. For example, thesubject is further administered an anti-CTLA-4 antibody, anti-PD-1,anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25,anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS,anti-TIGIT, and inhibitors of IDO. Blockade of CTLA-4 or PD-1/PD-L1 byantibodies can enhance the immune response to cancerous cells in thepatient. In particular, CTLA-4 blockade has been shown effective whenfollowing a vaccination protocol.

The optimum amount of each peptide to be included in the vaccinecomposition and the optimum dosing regimen can be determined by oneskilled in the art without undue experimentation. For example, thepeptide or its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c, i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c,i.p. and i.v. For example, doses of between 1 and 500 mg 50 μg and 1.5mg, preferably 10 μg to 500 μg, of peptide or DNA may be given and willdepend from the respective peptide or DNA. Doses of this range weresuccessfully used in previous trials (Brunsvig P F, et al., CancerImmunol Immunother. 2006; 55(12): 1553-1564; M. Staehler, et al., ASCOmeeting 2007; Abstract No 3017). Other methods of administration of thevaccine composition are known to those skilled in the art.

The inventive vaccine may be compiled so that the selection, numberand/or amount of peptides present in the composition is/are tissue,cancer, and/or patient-specific. For instance, the exact selection ofpeptides can be guided by expression patterns of the parent proteins ina given tissue to avoid side effects. The selection may be dependent onthe specific type of cancer, the status of the disease, earliertreatment regimens, the immune status of the patient, and, of course,the HLA-haplotype of the patient. Furthermore, the vaccine according tothe invention can contain individualized components, according topersonal needs of the particular patient. Examples include varying theamounts of peptides according to the expression of the relatedneoantigen in the particular patient, unwanted side-effects due topersonal allergies or other treatments, and adjustments for secondarytreatments following a first round or scheme of treatment.

Such vaccines may be administered to an individual already sufferingfrom cancer. In therapeutic applications, such vaccines are administeredto a patient in an amount sufficient to elicit an effective CTL responseto the tumor antigen and to cure or at least partially arrest symptomsand/or complications. An amount adequate to accomplish this is definedas “therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally range for the initial immunization(that is for therapeutic or prophylactic administration) from about 1.0μg to about 50,000 μg of peptide for a 70 kg patient, followed byboosting dosages or from about 1.0 μg to about 10,000 μg of peptidepursuant to a boosting regimen over weeks to months depending upon thepatient's response and condition and possibly by measuring specific CTLactivity in the patient's blood. It should be kept in mind that thepeptide and compositions of the present invention may generally beemployed in serious disease states, that is, life-threatening orpotentially life threatening situations, especially when the cancer hasmetastasized. For therapeutic use, administration should begin as soonas possible after the detection or surgical removal of tumors. This isfollowed by boosting doses until at least symptoms are substantiallyabated and for a period thereafter. The pharmaceutical compositions(e.g., vaccine compositions) for therapeutic treatment are intended forparenteral, topical, nasal, oral or local administration. Preferably,the pharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thecompositions may be administered at the site of surgical excision toinduce a local immune response to the tumor.

Such embodiments are not limited to a particular type of adjuvant.Generally, adjuvants are any substance whose admixture into the vaccinecomposition increases or otherwise modifies the immune response to themutant peptide. Carriers are scaffold structures, for example apolypeptide or a polysaccharide, to which the antigenic peptide (e.g.,neo-antigenic peptide) is capable of being associated. Optionally,adjuvants are conjugated covalently or non-covalently to the peptides orpolypeptides of the invention.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedreaction, or reduction in disease symptoms. For example, an increase inhumoral immunity is typically manifested by a significant increase inthe titer of antibodies raised to the antigen, and an increase in T-cellactivity is typically manifested in increased cell proliferation, orcellular cytotoxicity, or cytokine secretion. An adjuvant may also alteran immune response, for example, by changing a primarily humoral or Th2response into a primarily cellular, or Th1 response.

Suitable adjuvants include, but are not limited to 1018 ISS, aluminumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF,IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles,resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (AquilaBiotech, Worcester, Mass., USA) which is derived from saponin,mycobacterial extracts and synthetic bacterial cell wall mimics, andother proprietary adjuvants such as Ribi's Detox. Quil or Superfos.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Dupuis M, etal., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand.1998; 92:3-11). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen -presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J ImmunotherEmphasis Tumor Immunol. 1996 (6):414-418). Toll like receptors (TLRs)may also be used as adjuvants, and are important members of the familyof pattern recognition receptors (PRRs) which recognize conserved motifsshared by many micro-organisms, termed “pathogen-associated molecularpatterns” (PAMPS).

Recognition of these “danger signals” activates multiple elements of theinnate and adaptive immune system. TLRs are expressed by cells of theinnate and adaptive immune systems such as dendritic cells (DCs),macrophages, T and B cells, mast cells, and granulocytes and arelocalized in different cellular compartments, such as the plasmamembrane, lysosomes, endosomes, and endolysosomes. Different TLRsrecognize distinct PAMPS. For example, TLR4 is activated by LPScontained in bacterial cell walls, TLR9 is activated by unmethylatedbacterial or viral CpG DNA, and TLR3 is activated by double strandedRNA. TLR ligand binding leads to the activation of one or moreintracellular signaling pathways, ultimately resulting in the productionof many key molecules associated with inflammation and immunity(particularly the transcription factor NF-κB and the Type-Iinterferons). TLR mediated DC activation leads to enhanced DCactivation, phagocytosis, upregulation of activation and co-stimulationmarkers such as CD80, CD83, and CD86, expression of CCR7 allowingmigration of DC to draining lymph nodes and facilitating antigenpresentation to T cells, as well as increased secretion of cytokinessuch as type I interferons, IL-12, and IL-6. All of these downstreamevents are critical for the induction of an adaptive immune response.

Other receptors which may be targeted include the toll-like receptors(TLRs). TLRs recognize and bind to pathogen-associated molecularpatterns (PAMPs). PAMPs target the TLR on the surface of the dendriticcell and signals internally, thereby potentially increasing DC antigenuptake, maturation and T-cell stimulatory capacity. PAMPs conjugated tothe particle surface or co-encapsulated include unmethylated CpG DNA(bacterial), double-stranded RNA (viral), lipopolysacharride(bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial),zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial),flagellin (bacterial) poly(inosinic-cytidylic) acid (bacterial),lipoteichoic acid (bacterial) or imidazoquinolines (synthetic).

Among the most promising cancer vaccine adjuvants currently in clinicaldevelopment are the TLR9 agonist CpG and the synthetic double-strandedRNA (dsRNA) TLR3 ligand poly-ICLC. In preclinical studies poly-ICLCappears to be the most potent TLR adjuvant when compared to LPS and CpGdue to its induction of pro-inflammatory cytokines and lack ofstimulation of IL-10, as well as maintenance of high levels ofco-stimulatory molecules in DCs. Furthermore, poly-ICLC was recentlydirectly compared to CpG in non-human primates (rhesus macaques) asadjuvant for a protein vaccine consisting of human papillomavirus(HPV)16 capsomers (Stahl-Hennig C, Eisenblatter M, Jasny E, et al.Synthetic double-stranded RNAs are adjuvants for the induction of Thelper 1 and humoral immune responses to human papillomavirus in rhesusmacaques. PLoS pathogens. April 2009; 5(4)).

In some embodiments, the adjuvant is a dendritic cell targeting molecule(DC). DC is potent and is responsible for initiating antigen-specificimmune responses. One biological feature of DCs is their ability tosense conditions under which antigen is encountered, initiating aprocess of “DC maturation”. Using receptors for various microbial andinflammatory products, DCs respond to antigen exposure in different waysdepending on the nature of the pathogen (virus, bacteria, protozoan)encountered. This information is transmitted to T cells by alteredpatterns of cytokine release at the time of antigen presentation inlymph nodes, altering the type of T cell response elicited. Thus,targeting DCs provides the opportunity not only to quantitativelyenhance the delivery of antigen and antigen responses in general, but toqualitatively control the nature of the immune response depending on thedesired vaccination outcome.

Dendritic cells express a number of cell surface receptors that canmediate the endocytosis of bound antigen. Targeting exogenous antigensto internalizing surface molecules on systemically-distributed antigenpresenting cells facilitates uptake of antigens and thus overcomes amajor rate-limiting step in immunization and thus in vaccination.

Dendritic cell targeting molecules include monoclonal or polyclonalantibodies or fragments thereof that recognize and bind to epitopesdisplayed on the surface of dendritic cells. Dendritic cell targetingmolecules also include ligands which bind to a cell surface receptor ondendritic cells. One such receptor, the lectin DEC-205, has been used invitro and in mice to boost both humoral (antibody-based) and cellular(CD8 T cell) responses by 2-4 orders of magnitude (see, e.g., Hawiger,et al., J. Exp. Med., 194(6):769-79 (2001); Bonifaz, et al., J. Exp.Med., 196(12):1627-38 (2002); Bonifaz, et al., J. Exp. Med.,199(6):815-24 (2004)).

A variety of other endocytic receptors, including a mannose-specificlectin (mannose receptor) and IgG Fc receptors, have also been targetedin this way with similar enhancement of antigen presentation efficiency.Other suitable receptors which may be targeted include, but are notlimited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock proteinreceptors and scavenger receptors.

In some embodiments, the adjuvant is CpG. CpG immuno stimulatoryoligonucleotides have also been reported to enhance the effects ofadjuvants in a vaccine setting. Without being bound by theory, CpGoligonucleotides act by activating the innate (non-adaptive) immunesystem via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9activation enhances antigen-specific humoral and cellular responses to awide variety of antigens, including peptide or protein antigens, live orkilled viruses, dendritic cell vaccines, autologous cellular vaccinesand polysaccharide conjugates in both prophylactic and therapeuticvaccines. More importantly, it enhances dendritic cell maturation anddifferentiation, resulting in enhanced activation of Th1 cells andstrong cytotoxic T-lymphocyte (CTL) generation, even in the absence ofCD4 T-cell help. The Th1 bias induced by TLR9 stimulation is maintainedeven in the presence of vaccine adjuvants such as alum or incompleteFreund's adjuvant (IFA) that normally promote a Th2 bias. CpGoligonucleotides show even greater adjuvant activity when formulated orco-administered with other adjuvants or in formulations such asmicroparticles, nano particles, lipid emulsions or similar formulations,which are especially necessary for inducing a strong response when theantigen is relatively weak. They also accelerate the immune response andenabled the antigen doses to be reduced by approximately two orders ofmagnitude, with comparable antibody responses to the full-dose vaccinewithout CpG in some experiments (Arthur M. Krieg, Nature Reviews, DrugDiscovery, 5, Jun. 2006, 471-484). U.S. Pat. No. 6,406,705 B1 describesthe combined use of CpG oligonucleotides, non-nucleic acid adjuvants andan antigen to induce an antigen-specific immune response. A commerciallyavailable CpG TLR9 antagonist is dSLIM (double Stem LoopImmunomodulator) by Mologen (Berlin, GERMANY), which is a preferredcomponent of the pharmaceutical composition of the present invention.Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR9 may also be used.

Xanthenone derivatives such as, for example, Vadimezan or AsA404 (alsoknown as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)), may also beused as adjuvants according to embodiments of the invention.Alternatively, such derivatives may also be administered in parallel tothe vaccine of the invention, for example via systemic or intratumoraldelivery, to stimulate immunity at the tumor site. Without being boundby theory, it is believed that such xanthenone derivatives act bystimulating interferon (IFN) production via the stimulator of IFN geneISTING) receptor (see e.g., Conlon et al. (2013) Mouse, but not HumanSTING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid, Journal of Immunology, 190:5216-25and Kim et al. (2013) Anticancer Flavonoids are Mouse-Selective STINGAgonists, 8: 1396-1401). Other examples of useful adjuvants include, butare not limited to, chemically modified CpGs (e.g. CpR, Idera),Poly(I:C)(e.g. polyl:CI2U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171,ipilimumab, tremelimumab, and SC58175, which may act therapeuticallyand/or as an adjuvant. The amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan without undue experimentation.Additional adjuvants include colony-stimulating factors, such asGranulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).

Poly-ICLC is a synthetically prepared double-stranded RNA consisting ofpolyl and polyC strands of average length of about 5000 nucleotides,which has been stabilized to thermal denaturation and hydrolysis byserum nucleases by the addition of polylysine andcarboxymethylcellulose. The compound activates TLR3 and the RNAhelicase-domain of MDA5, both members of the PAMP family, leading to DCand natural killer (NK) cell activation and production of a “naturalmix” of type I interferons, cytokines, and chemokines. Furthermore,poly-ICLC exerts a more direct, broad host-targeted anti-infectious andpossibly antitumor effect mediated by the two IFN-inducible nuclearenzyme systems, the 2′ 5′-OAS and the Pl/eIF2a kinase, also known as thePKR (4-6), as well as RIG-I helicase and MDA5.

Such methods are not limited to generating sHDL nanoparticles associatedwith (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed,admixed) an antigen and an adjuvant (e.g., dendritic cell targetingmolecule). In some embodiments, the antigen and adjust are conjugated toouter surface of the sHDL nanoparticle.

In some embodiments, the sHDL nanoparticle is synthesized withthiol-reactive phospholipids that permit reduction-sensitive linkage ofthe antigen and/or adjuvant. In some embodiments, loading of the DCwithin the sHDL nanoparticle is facilitated through cholesterolmodification of the DC molecule. In some embodiments, lyophilizationmethods are used for the preparation of homogenous sHDL. In someembodiments, phospholipids and ApoA mimetic peptides are dissolved inglacial acetic acid and lyophilized. In some embodiments, antigenpeptides are incubated with sHDL in a buffer (e.g., a sodium phosphatebuffer (pH 7.4)) (e.g., at room temperature for 3 hours) to allow forthe conjugation of antigen peptides. In some embodiments, theunconjugated antigen peptides are removed using a desalting column(MWCO=7000 Da). In some embodiments, incorporation of the cholesterolmodified DC (Cho-DC) to sHDL involves incubation with sHDL at roomtemperature for approximately 30 min.

Such embodiments are not limited to a particular manner ofcharacterizing the sHDL conjugated with antigen and DC. In someembodiments, the morphology of sHDL is observed by TEM. In someembodiments, the size distribution of sHDL is analyzed by dynamic lightscattering (DLS) using a Malven Nanosizer instrument and GPC assay.

The sHDL nanoparticles configured to activate an immune response (e.g.,sHDL-αGalCer) (e.g., Ag/DC-sHDL) are useful for activating T cells insubjects for prophylactic and therapeutic applications. Activation of Tcells by nanoparticle vaccine compositions increases theirproliferation, cytokine production, differentiation, effector functionsand/or survival. Methods for measuring these are well known to those inthe art. The T cells activated by the nanoparticle vaccine compositionscan be any cell which express the T cell receptor, including α/β and γ/δT cell receptors. T-cells include all cells which express CD3, includingT-cell subsets which also express CD4 and CD8. T-cells include bothnaive and memory cells and effector cells such as CTL. T-cells alsoinclude regulatory cells such as Th1, Tc1, Th2, Tc2, Th3, Treg, and Tr1cells. T-cells also include NKT-cells and similar unique classes of theT-cell lineage. In some embodiments, the T cells that are activated areCD8⁺ T cells.

In general, compositions comprising the sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) areuseful for treating a subject having or being predisposed to any diseaseor disorder to which the subject's immune system mounts an immuneresponse. The compositions are useful as prophylactic vaccines, whichconfer resistance in a subject to subsequent exposure to infectiousagents. The compositions are also useful as therapeutic vaccines, whichcan be used to initiate or enhance a subject's immune response to apre-existing antigen, such as a tumor antigen in a subject with cancer,or a viral antigen in a subject infected with a virus. The compositionsare also useful as desensitizing vaccines, which function to “tolerize”an individual to an environmental antigen, such as an allergen.

The ability to target these compositions to professionalantigen-presenting cells such as dendritic cells, and the ability ofthese compositions to elicit T-cell mediated immune responses by causingcross-presentation of antigens makes these compositions especiallyuseful for eliciting a cell-mediated response to a disease-relatedantigen in order to attack the disease. Thus, in some embodiments, thetype of disease to be treated or prevented is a malignant tumor or achronic infectious disease caused by a bacterium, virus, protozoan,helminth, or other microbial pathogen that enters intracellularly and isattacked, i.e., by the cytotoxic T lymphocytes.

The desired outcome of a prophylactic, therapeutic or de-sensitizedimmune response may vary according to the disease, according toprinciples well known in the art. For example, an immune responseagainst an infectious agent may completely prevent colonization andreplication of an infectious agent, affecting “sterile immunity” and theabsence of any disease symptoms. However, a vaccine against infectiousagents may be considered effective if it reduces the number, severity orduration of symptoms; if it reduces the number of individuals in apopulation with symptoms; or reduces the transmission of an infectiousagent. Similarly, immune responses against cancer, allergens orinfectious agents may completely treat a disease, may alleviatesymptoms, or may be one facet in an overall therapeutic interventionagainst a disease. For example, the stimulation of an immune responseagainst a cancer may be coupled with surgical, chemotherapeutic,radiologic, hormonal and other immunologic approaches in order to affecttreatment.

Subjects with or exposed to infectious agents can be treatedtherapeutically or prophylactically the sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) asdisclosed herein. Infectious agents include bacteria, viruses andparasites. In some instances, the subject can be treatedprophylactically, such as when there may be a risk of developing diseasefrom an infectious agent. An individual traveling to or living in anarea of endemic infectious disease may be considered to be at risk and acandidate for prophylactic vaccination against the particular infectiousagent. Preventative treatment can be applied to any number of diseaseswhere there is a known relationship between the particular disease and aparticular risk factor, such as geographical location or workenvironment.

Subjects with or at risk for developing malignant tumors can be treatedtherapeutically or prophylactically the sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) asdisclosed herein. In a mature animal, a balance usually is maintainedbetween cell renewal and cell death in most organs and tissues. Thevarious types of mature cells in the body have a given life span; asthese cells die, new cells are generated by the proliferation anddifferentiation of various types of stem cells. Under normalcircumstances, the production of new cells is so regulated that thenumbers of any particular type of cell remain constant. Occasionally,though, cells arise that are no longer responsive to normalgrowth-control mechanisms. These cells give rise to clones of cells thatcan expand to a considerable size, producing a tumor or neoplasm. Atumor that is not capable of indefinite growth and does not invade thehealthy surrounding tissue extensively is benign. A tumor that continuesto grow and becomes progressively invasive is malignant. The term cancerrefers specifically to a malignant tumor. In addition to uncontrolledgrowth, malignant tumors exhibit metastasis. In this process, smallclusters of cancerous cells dislodge from a tumor, invade the blood orlymphatic vessels, and are carried to other tissues, where they continueto proliferate. In this way a primary tumor at one site can give rise toa secondary tumor at another site. The sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) asdisclosed herein are useful for treating subjects having malignanttumors.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands. Amelanoma is a type of carcinoma of the skin for which this invention isparticularly useful. Sarcomas, which arise less frequently, are derivedfrom mesodermal connective tissues such as bone, fat, and cartilage. Theleukemias and lymphomas are malignant tumors of hematopoietic cells ofthe bone marrow. Leukemias proliferate as single cells, whereaslymphomas tend to grow as tumor masses. Malignant tumors may show up atnumerous organs or tissues of the body to establish a cancer.

The types of cancer that can be treated in with the provided sHDLnanoparticles configured to activate an immune response (e.g.,sHDL-αGalCer) (e.g., Ag/DC-sHDL) include, but are not limited to, thefollowing: bladder, brain, breast, cervical, colo-rectal, esophageal,kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin,stomach, uterine, and the like. Administration is not limited to thetreatment of an existing tumor or infectious disease but can also beused to prevent or lower the risk of developing such diseases in anindividual, i.e., for prophylactic use. Potential candidates forprophylactic vaccination include individuals with a high risk ofdeveloping cancer, i.e., with a personal or familial history of certaintypes of cancer.

Subjects with or at risk for exposure to allergens can be treatedtherapeutically or prophylactically the sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) asdisclosed herein. Such sHDL nanoparticles may be administered tosubjects for the purpose of preventing and/or attenuating allergicreactions, such as allergic reactions which lead to anaphylaxis.Allergic reactions may be characterized by the T_(H)2 responses againstan antigen leading to the presence of IgE antibodies. Stimulation ofT_(H)1 immune responses and the production of IgG antibodies mayalleviate allergic disease. Thus, the sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) asdisclosed herein are useful for producing antibodies that prevent and/orattenuate allergic reactions in subjects exposed to allergens.

Subjects with or at risk for immunosuppressed conditions can be treatedtherapeutically or prophylactically the sHDL nanoparticles configured toactivate an immune response (e.g., sHDL-αGalCer) (e.g., Ag/DC-sHDL) asdisclosed herein. The sHDL nanoparticle vaccines disclosed herein can beused for treatment of disease conditions characterized byimmunosuppression, including, but not limited to, AIDS or AIDS-relatedcomplex, idiopathic immuno suppression, drug induced immunosuppression,other virally or environmentally-induced conditions, and certaincongenital immune deficiencies. Such sHDL nanoparticle vaccinecompositions can also be employed to increase immune function that hasbeen impaired by the use of radiotherapy of immunosuppressive drugs(e.g., certain chemotherapeutic agents), and therefore can beparticularly useful when used in conjunction with such drugs orradiotherapy.

Subjects with or at risk for coronary heart disease and/or elevatedLDL-C levels can be treated therapeutically or prophylactically the sHDLnanoparticles configured to activate an immune response as disclosedherein. While effectiveness of mAb therapy against PCSK9 has established(see, e.g., Banerjee, Y.; et al., New England Journal of Medicine 2012,366 (25), 2425-2426; Stein, E. A.; et al., Circulation 2013, 128 (19),2113-2120), development of more durable PCSK9 vaccines are needed. Inaddition, one of the challenges for PCSK9 vaccines is that selfantigens, such as PCSK9 peptides, are not immunogenic, unless they arecoupled to vaccine/adjuvant systems that can efficiently co-deliverantigens and immunostimulatory molecules to immune cells (see, e.g.,Krishnamachari, Y.; et al., Advanced Drug Delivery Reviews 2009, 61 (3),205-217; Hamdy, S.; et al., Advanced Drug Delivery Reviews 2011, 63(10-11), 943-955).

Embodiments of the present invention wherein sHDL nanoparticles areconjugated with a PCSK9-antigen and a CpG-adjuvant (PCSK9-Ag/CpG-sHDL)address such needs. Indeed, vaccination against PCSK9 withPCSK9-Ag/CpG-sHDL embodiments effectively inhibits interaction betweenPCSK9 and LDLR, while avoiding the need for repeated injections ofexpensive mAb (see, e.g., Fattori, E.; et al., Journal of Lipid Research2012, 53 (8), 1654-1661; Gergana Galabova, et al., PLOS ONE 2014, 9(12)). Moreover, such PCSK9-Ag/CpG-sHDL nanoparticles have asufficiently small size (e.g., 10-45 nm) permitting efficient drainageto the lymph nodes compared to larger particles (see, e.g., Bachmann, M.F.; et al., Nature Reviews Immunology 2010, 10 (11), 787-796).

In general, methods of administering vaccines as disclosed herein (e.g.,sHDL nanoparticles configured to activate an immune response (e.g.,sHDL-αGalCer) (e.g., Ag/DC-sHDL)) are well known in the art. Anyacceptable method known to one of ordinary skill in the art may be usedto administer a formulation to the subject. The administration may belocalized (i.e., to a particular region, physiological system, tissue,organ, or cell type) or systemic. Vaccines can be administered by anumber of routes including, but not limited to: oral, inhalation (nasalor pulmonary), intravenous, intraperitoneal, intramuscular, transdermal,subcutaneous, topical, sublingual, or rectal means. Injections can bee.g., intravenous, intradermal, subcutaneous, intramuscular, orintraperitoneal. In some embodiments, the injections can be given atmultiple locations.

Administration of the formulations may be accomplished by any acceptablemethod which allows an effective amount of the vaccine to reach itstarget. The particular mode selected will depend upon factors such asthe particular formulation, the severity of the state of the subjectbeing treated, and the dosage required to induce an effective immuneresponse. As generally used herein, an “effective amount” is that amountwhich is able to induce an immune response in the treated subject. Theactual effective amounts of vaccine can vary according to the specificantigen or combination thereof being utilized, the particularcomposition formulated, the mode of administration, and the age, weight,condition of the individual being vaccinated, as well as the route ofadministration and the disease or disorder.

In certain embodiments, glycolipids encapsulated within sHDLnanoparticles are used as stimulators of natural killer T cell-mediatedimmune responses.

Natural killer T (NKT) cells are a heterogeneous group of T cells thatshare properties of both T cells and natural killer cells. Many of thesecells recognize the non-polymorphic CD1d molecule, an antigen-presentingmolecule that binds self and foreign lipids and glycolipids. NKT cellsconstitute only approximately 0.1% of all peripheral blood T cells. NKTcells are a subset of T cells that coexpress an αβ T-cell receptor, butalso express a variety of molecular markers that are typicallyassociated with NK cells, such as NK1.1. The best-known NKT cells differfrom conventional αβ T cells in that their T-cell receptors are far morelimited in diversity (‘invariant’ or ‘type 1’ NKT). They and otherCD1d-restricted T cells (‘type 2’ NKT) recognize lipids and glycolipidspresented by CD1d molecules, a member of the CD1 family ofantigen-presenting molecules, rather than peptide-majorhistocompatibility complexes (MHCs). NKT cells include both NK1.1⁺ andNK1.1⁻, as well as CD4⁺, CD4⁻, CD8⁺ and CD8⁻ cells.

In some embodiments the glycolipid is the synthetic glycolipidalpha-galactosylceramide (αGalCer). Dendritic cells presenting antigensin the context of CD1d can lead to rapid innate and prolonged productionof cytokines such as interferon and IL-4 by natural killer T cells (NKTcells). CD1d is a major histocompatibility complex class I-like moleculethat presents glycolipid antigens to a subset of NKT cells.Advantageously, αGalCer is not toxic to humans and has been shown to actas an adjuvant, priming both antigen-specific CD4+ and CD8+ T cellresponses. For example, it has been shown that αGalCer in conjunctionwith a malaria vaccine can lead to cytotoxic responses against infectedcells, which is an ideal scenario for vaccines against infectiousdiseases. In addition to αGalCer, other glycolipids that function asadjuvants to activate NKT cell-mediated immune responses can be used.

The present invention is not limited to particular methods forgenerating sHDL nanoparticles having encapsulated αGalCer. For example,in some embodiments, lyophilization methods are used for the preparationof homogenous sHDL. In some embodiments, phospholipids and ApoA mimeticpeptides are dissolved in glacial acetic acid and lyophilized. In someembodiments, loading of αGalCer into the sHDL nanoparticle isfacilitated through hydrophobic interactions between the αGalCer and thesHDL. In some embodiments, the lyophilized phospholipids and ApoAmimetic peptides are hydrated (e.g., hydrated in PBS (pH 7.4)) andthermocycled above and below the transition temperature (Tm) ofphospholipids to form blank sHDL, which are next incubated with αGalCerat room temperature for an optimal amount of time (e.g., 5, 10, 20, 25,30, 35, 50, 80, 120, 360 minutes) to form sHDL comprising encapsulatedαGalCer.

Such embodiments are not limited to a particular manner ofcharacterizing the sHDL comprising encapsulated αGalCer. In someembodiments, the morphology of sHDL-αGalCer is observed by TEM. In someembodiments, the size distribution of sHDL-αGalCer is analyzed bydynamic light scattering (DLS) using a Malven Nanosizer instrument andGPC assay.

Such embodiments are not limited to a particular manner of assessing thedelivery profile of the αGalCer in vitro and in vivo. In someembodiments, labelling the molecules with an imaging agent (e.g.,fluorescent dye Cy3) permits visualization of the biodistribution ofαGalCer molecules at the organ level and also the intracellular deliveryprofile.

In certain embodiments, the present invention provides methods forinducing a natural killer T cell-mediated immune response in a cellcomprising exposing the cell to a composition comprising an αGalCerglycolipid encapsulated within a sHDL nanoparticle, wherein suchexposure results in the induction of a natural killer T cell-mediatedimmune response. In some embodiments, the cells are in vivo cells. Insome embodiments, the cells are in vitro cells. In some embodiments, thecells are ex vivo cells.

In certain embodiments, the present invention provides methods forinducing a natural killer T cell-mediated immune response in a subject(e.g., a human patient) comprising administering to the patient apharmaceutical composition comprising an αGalCer glycolipid encapsulatedwithin a sHDL nanoparticle, wherein such administration results in theinduction of a natural killer T cell-mediated immune response.

Additional Embodiments

In certain embodiments, the sHDL nanoparticles as described herein(e.g., configured for RNA Interference) (e.g., configured for activatingan immune response) are associated with (e.g., complexed, conjugated,encapsulated, absorbed, adsorbed, admixed) one or more therapeuticagents. Such embodiments are not limited to particular type or kind oftherapeutic agent.

In some embodiments, the therapeutic agent configured for treatingand/or preventing cancer. Examples of such therapeutic agents include,but are not limited to, chemotherapeutic agents, anti-oncogenic agents,anti-angiogenic agents, tumor suppressor agents, anti-microbial agents,etc.

In some embodiments, the therapeutic agent is configured for treatingand/or preventing autoimmune disorders and/or inflammatory disorders.Examples of such therapeutic agents include, but are not limited to,disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate,sulfasalazine, hydroxychloroquine), biologic agents (e.g., rituximab,infliximab, etanercept, adalimumab, golimumab), nonsteroidalanti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen,naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen,tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids(e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g.,adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1inhibitors, and metalloprotease inhibitors. In some embodiments, thetherapeutic agents include, but are not limited to, infliximab,adalimumab, etanercept, parenteral gold or oral gold.

In some embodiments, the therapeutic agent is configured for treatingand/or preventing cardiovascular related disorders (e.g.,atherosclerosis, heart failure, arrhythmia, atrial fibrillation,hypertension, coronary artery disease, angina pectoris, etc.). Examplesof therapeutic agents known to be useful in treating and/or preventingcardiovascular related disorders include, angiotensin-converting enzyme(ACE) inhibitors (e.g., benazepril, enalapril, Lisinopril, perindopril,Ramipril), adenosine, alpha blockers (alpha adrenergic antagonistmedications) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine,terazosin, doxazosin, guanfacine, methyldopa, prazosin), angtiotensin IIreceptor blockers (ARBs) (e.g., candesartan, irbesartan, olmesartanmedoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan),antiocoagulants (e.g., heparin fondaparinux, warfarin, ardeparin,enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplateletagents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine,cilostazol, dipyridamole, sulfinpyrazone, tirofiban), beta blockers(e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol,propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol),calcium channel blockers (e.g., amlopidine, felodipine, isradipine,nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine),diuretics, aldosterone blockers, loop diuretics (e.g., bumetanide,furosemide, ethacrynic acid, torsemide), potassium-sparing diuretics,thiazide diuretics (e.g., chlorothiazide, chlorthalidone,hydrochlorothiazide, hydroflumethiazide, methyclothiazide, metolazone,polythiazide, quinethazone, trichlormethiazide), inoptropics, bile acidsequestrants (e.g., cholestyramine, coletipol, colesevelam), fibrates(e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g.,atorvastatinm, lovastatin, simvastatin, fluvastatin, pravastatin),selective cholesterol absorption inhibitors (e.g., ezetimibe), potassiumchannel blockers (e.g., amidarone, ibutilide, dofetilide), sodiumchannel blockers (e.g., disopyramide, mexiletine, procainamide,quinidine, flecainide, moricizine, propafenone), thrombolytic agents(e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase,urokinase), vasoconstrictors, vasodilators (e.g., hydralazine,minoxidil, mecamylamine, isorbide dintrate, isorbide mononitrate,nitroglycerin).

Generally, the sHDL nanoparticles so formed are spherical and have adiameter of from about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, thesHDL nanoparticles are subjected to size exclusion chromatography toyield a more homogeneous preparation.

In some embodiments, the sHDL nanoparticles are further associated with(e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, admixed)agents useful for determining the location of administered particles.Agents useful for this purpose include fluorescent tags, radionuclidesand contrast agents.

Suitable imaging agents include, but are not limited to, fluorescentmolecules such as those described by Molecular Probes (Handbook offluorescent probes and research products), such as Rhodamine,fluorescein, Texas red, Acridine Orange, Alexa Fluor (various),Allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various),Calcien, Calcium Crimson, Calcium green, Calcium Orange,6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DID,Di1, DiO, DiR, ELF 97, Eosin, ER Tracker Blue-White, EthD-1, Ethidiumbromide, Fluo-3, Fluo4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258,Hoechst 33342, 7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye,Lissamine rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167,LysoSensor Green, LysoSensor Yellow/Blu, Lysotracker Green FM, MagnesiumGreen, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos,MitoTracker Red CMXRos, Monobromobimane, NBD amines, NeruoTrace 500/525green, Nile red, Oregon Green, Pacific Blue. POP-1, Propidium iodide,Rhodamine 110, Rhodamine Red, R-Phycoerythrin, Resorfin, RH414, Rhod-2,Rhodamine Green, Rhodamine 123, ROX dye, Sodium Green, SYTO blue(various), SYTO green (Various), SYTO orange (various), SYTOX blue,SYTOX green, SYTOX orange, Tetramethylrhodamine B, TOT-1, TOT-3,X-rhod-1, YOYO-1, YOYO-3. In some embodiments, ceramides are provided asimaging agents. In some embodiments, S1P agonists are provided asimaging agents.

Additionally radionuclides can be used as imaging agents. Suitableradionuclides include, but are not limited to radioactive species ofFe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) Indium, Gallium andTechnetium. Other suitable contrast agents include metal ions generallyused for chelation in paramagnetic T1-type MIR contrast agents, andinclude di- and tri-valent cations such as copper, chromium, iron,gadolinium, manganese, erbium, europium, dysprosium and holmium. Metalions that can be chelated and used for radionuclide imaging, include,but are not limited to metals such as gallium, germanium, cobalt,calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium,technetium, rhenium, platinum, thallium and samarium. Additionally metalions known to be useful in neutron-capture radiation therapy includeboron and other metals with large nuclear cross-sections. Also suitableare metal ions useful in ultrasound contrast, and X-ray contrastcompositions.

Examples of other suitable contrast agents include gases or gas emittingcompounds, which are radioopaque.

In some embodiments, the sHDL nanoparticles are further associated with(e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, admixed)a targeting agent. In some embodiments, targeting agents are used toassist in delivery of the sHDL-TA nanoparticles to desired body regions(e.g., bodily regions affected by a cardiovascular related disorder).Examples of targeting agents include, but are not limited to, anantibody, receptor ligand, hormone, vitamin, and antigen, however, thepresent invention is not limited by the nature of the targeting agent.In some embodiments, the antibody is specific for a disease-specificantigen. In some embodiments, the receptor ligand includes, but is notlimited to, a ligand for CFTR, EGFR, estrogen receptor, FGR2, folatereceptor, IL-2 receptor, glycoprotein, and VEGFR. In some embodiments,the receptor ligand is folic acid.

In some embodiments, the sHDL nanoparticles of the present invention maybe delivered to local sites in a patient by a medical device. Medicaldevices that are suitable for use in the present invention include knowndevices for the localized delivery of therapeutic agents. Such devicesinclude, but are not limited to, catheters such as injection catheters,balloon catheters, double balloon catheters, microporous ballooncatheters, channel balloon catheters, infusion catheters, perfusioncatheters, etc., which are, for example, coated with the therapeuticagents or through which the agents are administered; needle injectiondevices such as hypodermic needles and needle injection catheters;needleless injection devices such as jet injectors; coated stents,bifurcated stents, vascular grafts, stent grafts, etc.; and coatedvaso-occlusive devices such as wire coils.

Exemplary devices are described in U.S. Pat. Nos. 5,935,114; 5,908,413;5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719;5,851,228; 5,843,089; 5,800,519; 5,800,508; 5,800,391; 5,354,308;5,755,722; 5,733,303; 5,866,561; 5,857,998; 5,843,003; and 5,933,145;the entire contents of which are incorporated herein by reference.Exemplary stents that are commercially available and may be used in thepresent application include the RADIUS (SCIMED LIFE SYSTEMS, Inc.), theSYMPHONY (Boston Scientific Corporation), the Wallstent (SchneiderInc.), the PRECEDENT II (Boston Scientific Corporation) and the NIR(Medinol Inc.). Such devices are delivered to and/or implanted at targetlocations within the body by known techniques.

In some embodiments, the present invention also provides kits comprisingsHDL nanoparticles as described herein. In some embodiments, the kitscomprise one or more of the reagents and tools necessary to generatesHDL nanoparticles, and methods of using such sHDL nanoparticles.

The sHDL nanoparticles of the present invention may be characterized forsize and uniformity by any suitable analytical techniques. Theseinclude, but are not limited to, atomic force microscopy (AFM),electrospray-ionization mass spectroscopy, MALDI-TOF mass spectroscopy,¹³C nuclear magentic resonance spectroscopy, high performance liquidchromatography (HPLC) size exclusion chromatography (SEC) (equipped withmulti-angle laser light scattering, dual UV and refractive indexdetectors), capillary electrophoresis and get electrophoresis. Theseanalytical methods assure the uniformity of the sHDL nanoparticlepopulation and are important in the production quality control foreventual use in in vivo applications.

In some embodiments, gel permeation chromatography (GPC), which canseparate sHDL nanoparticles from liposomes and free ApoA-I mimeticpeptide, is used to analyze the sHDL-TA nanoparticles. In someembodiments, the size distribution and zeta-potential is determined bydynamic light scattering (DLS) using, for example, a Malven Nanosizerinstrument.

Where clinical applications are contemplated, in some embodiments of thepresent invention, the sHDL nanoparticles are prepared as part of apharmaceutical composition in a form appropriate for the intendedapplication. Generally, this entails preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals. However, in some embodiments of thepresent invention, a straight sHDL nanoparticle formulation may beadministered using one or more of the routes described herein.

In preferred embodiments, the sHDL nanoparticles are used in conjunctionwith appropriate salts and buffers to render delivery of thecompositions in a stable manner to allow for uptake by target cells.Buffers also are employed when the sHDL nanoparticles are introducedinto a patient. Aqueous compositions comprise an effective amount of thesHDL nanoparticles to cells dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. Except insofar as any conventional media or agent is incompatiblewith the vectors or cells of the present invention, its use intherapeutic compositions is contemplated. Supplementary activeingredients may also be incorporated into the compositions.

In some embodiments of the present invention, the active compositionsinclude classic pharmaceutical preparations. Administration of thesecompositions according to the present invention is via any common routeso long as the target tissue is available via that route. This includesoral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection.

The active sHDL nanoparticles may also be administered parenterally orintraperitoneally or intratumorally. Solutions of the active compoundsas free base or pharmacologically acceptable salts are prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The carrier may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial anantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it may be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activesHDL nanoparticles in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, sHDL nanoparticles are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution is suitably buffered, if necessary,and the liquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). In some embodiments of the present invention, the activeparticles or agents are formulated within a therapeutic mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses may be administered.

Additional formulations that are suitable for other modes ofadministration include vaginal suppositories and pessaries. A rectalpessary or suppository may also be used. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or the urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries areusually globular or oviform and weighing about 5 g each. Vaginalmedications are available in a variety of physical forms, e.g., creams,gels or liquids, which depart from the classical concept ofsuppositories. The sHDL nanoparticles also may be formulated asinhalants.

The present invention also includes methods involving co-administrationof the sHDL nanoparticles as described herein with one or moreadditional active agents. Indeed, it is a further aspect of thisinvention to provide methods for enhancing prior art therapies and/orpharmaceutical compositions by co-administering the sHDL nanoparticlesof this invention. In co-administration procedures, the agents may beadministered concurrently or sequentially. In some embodiments, the sHDLnanoparticles described herein are administered prior to the otheractive agent(s). The agent or agents to be co-administered depends onthe type of condition being treated.

The present disclosure further provides kits comprising compositionscomprising sHDL nanoparticles as described herein or the ingredientsnecessary to synthesize the sHDL nanoparticles as described herein. Insome embodiments, the kit includes all of the components necessary,sufficient or useful for administering such sHDL nanoparticles.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

EXAMPLE 1

This example describes the materials and methods for synthesis of a sHDLloaded with biomacromolecules

Materials

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and rhodamine(Rhod)-labeled DOPE (DOPE-Rhod) were all purchased form Avanti PolarLipids (Alabaster, AL).Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP) was additionally synthesized. All peptidesincluding HDL mimicking peptide (22A; SEQ ID NO: 4), SIINFEKL (SEQ IDNO: 385), CSSSIINFEKL (SEQ ID NO: 384), and FITC labeled CSSSIINFEK(SEQID NO:386) (FITC)L used were customized from GenScript. Theoligodeoxynucleotide TLR 9 ligand CpG 1826(5′-tccatgacgttcctgacgtt-3′(SEQ ID NO:382), lower case letters representphosphorothioate backbone) (SEQ ID NO: 343) and cholesterol modified CpG1826 (5′-tccatgacgttcctgacgtt-3′(SEQ ID NO:382)-TEG-cholesterol) wereordered from Intregrated DNA Technologies. HPLC grade solvents such asmethanol and acetonitrile were purchased from fisher scientific. Fetalbovine serum (FBS), penicillin-streptomycin, β-mercaptoethanol and ACKlysing buffer were purchased from Life Technologies (Grand Island,N.Y.). Granulocyte macrophage colony stimulating factor (GM-CSF) was theproduct of PeproTech (Rocky Hill, N.J.). Rat anti-mouse CD16/32,CD86-PE, CD40-APC, SIINFEKL H-2K^(b)-PE and MHC Class II-FITC were fromeBioscience (San Diego, Calif.). Rat anti-mouse CD8-APC, hamsteranti-mouse CD11c-PE and streptavidin-Cy7 were from BD Bioscience (SanJose, Calif.). iTAg tetramer/PE-H-2 Kb OVA (SIINFEKL) was purchased fromBeckman Coulter (Brea, Calif.).

Preparation of sHDL Nanoparticles Loaded with Peptides, Nucleic Acids,or Glycolipids.

DMPC and DOPE-PDP (weight ratio=4:0.25) were dissolved in chloroform.The mixture was dried with nitrogen flow for 5 min and then put in avacuum oven for 1 h. The obtained lipid film was hydrated in 10 mMsodium phosphate buffer (0.3117 g/L NaH₂PO₄.H₂O and 2.0747 g/LNa₂HPO₄.7H₂O) and sonicated with a bath sonicator for 10 min, followedby probe sonication for another 2.5 min. 22A peptide dissolved inendotoxin free water was added to the above mixture (22A:lipids=1:2,weight ratio), which was then subjected to heating (50° C.) for 3 minand cooling (ice water) for 3 min, with 3 cycles in total, to obtainsHDL.

To load tumor antigen peptides to sHDL, cysteine terminated tumorantigen peptides dissolved in endotoxin free water were added to theabove sHDL (antigen peptides:DOPE-PDP=2.5:1, molar ratio) and incubatedat room temperature with gentle shaking on an orbital shaker for 3 h.Unreacted tumor antigen peptides were removed by using Zeba SpinDesalting columns with a MWCO=7000 Da cutoff (Pierce) following themanufacturer's instructions. The conjugation efficiency of tumor antigenpeptides was calculated based on the decrease of DOPE-PDP determined bythe HPLC. Briefly, 200 μl sHDL formulations were freeze-dried andreconstituted in 300 μl methanol. The mixture was filtered by a 220 nmPTFE filter before 20 μl was injected to a Shimadzu HPLC system equippedwith a Vydac 219TP Diphenyl column (4.6 mm×250 mm ID). The two solventsused for the HPLC analysis consisted of water:trifluoroaceticacid=100:0.5 (mobile phase A) and methanol: acetonitrile:trifluoroaceticacid=50:50:0.05 (mobile phase B). Gradient programming of the solventsystem was: 25% mobile phase B was linearly increased to 100% B over 75min, linearly decreased to 25% B at 80 min, and maintained at 25% during80-90 min for equilibration before the next analysis. The flow rate was1 mL/min and the detection wavelength was 220 nm. The loading efficiencyof tumor antigen peptides was also determined by using FITC-labeledpeptides and measuring the fluorescence intensity of sHDL formulationsat Ex=490 nm and Em=520 nm after dissolving the formulations with 1%Triton X-100 containing PBS.

To load CpG to sHDL, different concentrations of cholesterol modifiedCpG (Cho-CpG) were incubated with sHDL at room temperature with gentleshaking on an orbital shaker for 30 min. The amount of CpG incorporatedinto sHDL and free CpG were analyzed by the gel permeationchromatography (GPC). Briefly, the sHDL formulations were diluted by PBSto a concentration of 0.5 mg/mL 22A peptide. The formulations werefiltered through a 220 nm filter before 40 μl samples were injected to aShimadzu HPLC system equipped with a TSKgel G2000SWx1 column (7.8 mmID×30 cm, Tosoh Bioscience LLC). The flow rate of mobile phase PBS (pH7.4) was set at 0.7 mL/min and detection wavelength was set at 260 nmfor CpG.

To load alpha-galactosylceramide (aGC) to sHDL, a lyophilization-basedmethod of producing sHDL was developed. Briefly, phospholipids, aGC andApoA mimetic peptides were dissolved in glacial acetic acid andlyophilized. The obtained powder was hydrated in PBS (pH 7.4) and cycledabove and below the transition temperature (Tm) of phospholipids to formaGC-sHDL. Similar protocol was utilized for loading siRNA into sHDL.Cholesterol-modified PCSK9 siRNA was incubated with blank sHDL at roomtemperature for 30 min to form PCSK9 siRNA-sHDL.

Morphology and Size Measurement of sHDL

The sHDL formulations were diluted to 0.5 mg/mL 22A with PBS and thesizes were measured by dynamic light scattering (DLS, Zetasizer NanoZSP, Malvern, UK). The morphology of sHDL was observed by transmissionelectron microscopy (TEM) after proper dilution of the original samples.

Preparation of Bone Marrow-Derived Dendritic Cells (BMDCs)

BMDCs were prepared. Briefly, femur and tibia of a mouse were harvested,washed and grinded in BMDC culture media (RPMI 1640 supplemented with10% FBS, 1% penicillin-streptomycin, 50 μM β-mercaptoethanol, and 20ng/ml GM-CSF). Cells were collected by passing the cell suspensionthrough a cell strainer (mesh size=40 μm), followed by centrifugation.Cells were seeded into non-tissue culture treated petri-dish at adensity of 2×10⁵ cells/ml, cultured at 37° C. with 5% CO₂. Culture mediawere refreshed on days 3, 6 and 8, and BMDCs were used during day 8-12.

Up-Regulation of Activation Markers on BMDCs

Immature BMDCs were plated at 1×10⁶ cells/well in 12-well plates 24 hprior to use. The old media were aspirated and BMDCs were washed oncewith PBS before incubated with 0.5 μg/mL different CpG-containingformulations or 0.5 μg/mL LPS (positive control) for 24 h at 37° C.BMDCs were harvested, washed once with FACS buffer (1% BSA in PBS),incubated with anti-CD16/32 at room temperature for 10 min, and thenstained with fluorescent probe-labeled antibodies against CD11c, CD40,CD80, CD86, and MHC class II at room temperature for 30 min. Finally,cells were washed twice by FACS buffer and resuspended in 2 μg/ml DAPIsolution and analyzed by flow cytometry (Cyan 5, Beckman Coulter, USA).

Antigen Presentation by BMDCs

Immature BMDCs were plated at 1×10⁶ cells/well in 12-well plates 24 hprior to use. The old media were aspirated and BMDCs were washed oncewith PBS before incubated with 0.5 μg/mL CpG and/or 0.5 μg/mL antigenpeptide-containing formulations in complete media for different lengthsof time (2, 6, 24, and 48 h) at 37° C. BMDCs were harvested, washed oncewith FACS buffer, incubated with anti-CD16/32 at room temperature for 10min, and then stained with PE-tagged anti mouse SIINFEKL H-2K^(b)monoclonal antibody 25-D1.16 at room temperature for 30 min. Finally,cells were washed twice with FACS buffer and resuspended in 2 μg/ml DAPIsolution and analyzed by flow cytometry (Cyan 5, Beckman Coulter, USA).

Imaging the Intracellular Delivery of sHDL-Based Peptide Vaccine withCLSM

1×10⁶ cells JAWSII cells in 2 mL complete media were seeded in 35 mmpetri dishes (MatTek) that have been pre-equilibrated with the sameculture media and allowed to settle overnight. To learn theintracellular delivery profile of sHDL itself, DOPE-Rhod was used tolabel the lipid of sHDL, and 22A peptide of sHDL was labeled byincubating sHDL with Texas Red®-X, Succinimidyl Ester (LifeTechnologies), followed by passing through the desalting column toremove the unreacted dye. These labeled sHDL were incubated with JAWSIIcells at 37° C. for 24 h. After incubation, cells were washed 3 timeswith PBS and the incubated with phenol and serum free media containing500 nM LysoTracker® Green DND-26 (Life Technologies) and 2 ug/mL Hoechstfor 30 min at 37° C. to stain the lysosomes and nuclei, respectively,before imaging using a confocal microscope (Nikon A1). To learn theintracellular delivery profile of the antigen peptides, freeCSSSIINFEK(SEQ ID NO:386) (FITC)L+CpG or sHDL-CSSSIINFEK(SEQ ID NO:386)(FITC)L/CpG were incubated with JAWSII cells for different lengths oftime (6, 24, and 48 h). After incubation, cells were washed 3 times withPBS and the incubated with phenol and serum free media containing 50 nMLYSOTRACKER Red DND-99 (Life Technologies) and 2 ug/mL Hoechst for 30min at 37° C. to stain the lysosomes and nuclei, respectively, beforeimaging using a confocal microscope (Nikon A1).

B3Z T Cell Activation In Vitro

BMDCs were plated at 5×10⁴ cells/well in a U-bottom 96-well plate andallowed to grow overnight. The old media were aspirated and BMDCs werewashed once with PBS before incubated with different concentrations(0.02, 0.1, and 0.5 μg/mL) of SIINFEKL and CpG containing formulationsfor 24 h or 48 h at 37° C. After incubation, cells were carefully washed3 times with PBS, and 10×10⁴ B3Z T cells/well were added and coculturedfor another 24 h in RPMI 1640 supplemented with 10% FBS, 2 mML-glutamine, 55 μM β-mercaptoethanol, 1 mM pyruvate and 100 U/mLpenicillin and 100 μg/mL streptomycin. Cells were then pelleted viacentrifugation (1500 rcf, 7 min). The media were carefully aspirated,and 150 μL CPRG/lysis buffer (0.15 mM chlorophenolred-β-D-galactopyranoside (CPRG), 0.1% Triton-X 100, 9 mM MgCl2, 100 uMmercaptoethanol in PBS) was added. The plates were incubated at 37° C.in the dark for 90 min, after which the absorbance of releasedchlorophenol red was measured at 570 nm using a plate reader.

Lymph Nodes Draining of Antigen Peptides

sHDL-CSSSIINFEK(SEQ ID NO:386)(FITC)L was prepared as described above.Female C57BL/6 mice of age 6-8 weeks were purchased from HarlanLaboratories. C57BL/6 mice were subcutaneously injected with freeCSSSIINFEK(SEQ ID NO:386) (FITC)L or sHDL-CSSSIINFEK(FITC)L(SEQ IDNO:386). 24 hours after injection, mice were euthanized by carbondioxide inhalation and axillary lymph nodes and inguinal lymph nodeswere harvested and imaged with IVIS optical imaging system (CaliperLifesciences).

In Vivo Vaccination and Analysis of Cytotoxic T Cell Responses inProphylactic and Therapeutic Settings of Melanoma Tumor Growth

C57BL/6 mice were immunized with different formulations containingSIINFEKL (15 μg/mouse) and CpG (15 μg/mouse) by subcutaneous injectionat the tail base following the predetermined schedule. The percent oftumor antigen specific CD8+ T cells were determined 7 days after eachvaccination by the tetramer staining assay. In brief, 100 μl of bloodwill be drawn from each mouse and the blood samples were lysed with ACKlysing buffer, followed by centrifugation to collect pellets, which werethen washed once by FACS buffer and blocked by CD16/32 blocking antibodyand incubated with PE labeled SIINFEKL tetramer for 30 min at roomtemperature. Samples were then incubated with anti-CD8-APC for 20 min onice. Cells were washed twice with FACS buffer and resuspended in 2 μg/mlDAPI solution for analysis by flow cytometry (Cyan 5, Beckman Coulter,USA). To examine the effect of T cell responses against tumor growth,one day after the last tetramer staining, the mice were challenged bysubcutaneous injection of 0.2 million B16.OVA/mouse on the right flank.The tumor development was monitored every other day and the tumor volumewas calculated by the following equation: tumorvolume=length×width²×0.52. In order to examine the effect of sHDLvaccination against established tumor, C57BL/6 mice were inoculated with0.2 million B16.OVA/mouse on the right flank by subcutaneous injectionon day 0. On day 4 and 11, the mice were immunized with differentformulations containing tumor antigen peptides (15 μg/mouse) and CpG (15μg/mouse). The percent of tumor antigen specific CD8+ T cells weredetermined on day 10 and 17 by the tetramer staining assay as describedabove. The tumor volume was monitored every other day.

aGC-CD1d Presentation Assay

JAWSII cells were seeded at a density of 0.2 million/well to 12-wellplates. After 48 h, media were replaced with fresh media containing 2000ng/mL of different formulations of aGC. After 20-24 h incubation withformulations, cells were harvested into FACS tubes by trypsination,washed twice by FACS buffer and then incubated with CD16/32 blockingreagent for 10 min at R.T. Cells were then incubated with anti-mouseaGC-CD1d-PE for 30 min at R.T, washed twice by FACS buffer, andsuspended in 0.3 mL FACS buffer containing DAPI for flow cytometry.

Characterization of PCSK9 siRNA-Loaded sHDL

To quantify the amount of PCSK9 siRNA molecules that are loaded intosHDL, various concentrations of PCSK9 siRNA was incubated with sHDL, andthe concentration of PCSK9 siRNA associated with sHDL versus free formwill be measured at 260 nm using the gel permeation chromatography (GPC)assay.

PCSK9 Knockdown in HepG2 Cells

Different formulations of PCSK9 siRNA were incubated with HepG2 cellsfor 48 h. After incubation, cells were washed twice with PBS and thecell lysate was prepared. The PCSK9 protein level was analyzed by thewestern blot assay.

Biodistribution of sHDL

To study the biodistribution of sHDL, DiD-loaded sHDL was intravenouslyinjected to the C57BL/6 mice. 24 h post injection, the mice wereeuthanized and the distribution of sHDL in major organs (heart, liver,spleen, lung and kidney) was analyzed using the IVIS optical imagingsystem.

EXAMPLE II

This example demonstrates that PCSK9 siRNA incorporated into sHDL canefficiently accumulate in the liver, deliver its cargo into SR-BIpositive cells, and knockdown PCSK9 in HepG2 cells. Rapid and cheaplyophilization methods for the preparation of homogeneous sHDLnanoparticles were implemented. The homogeneity of the sHDL wasconfirmed by transmission electron microscopy (TEM), dynamic laserscattering (DLS), and gel permeation chromatography (GPC) (FIG. 1A).When sHDL was labeled by the fluorescent dye DiR and intravenouslyinjected to mice, the majority of DiR signal was detected in the liver,with little or no signal in other organs (FIG. 1B). sHDL alsoefficiently delivered the fluorescent dye DiO into SR-BI positive cells(BHK-SR-BI), but not SR-BI negative cells (BHK-vector), and the uptakeby SR-BI positive cells was blocked by the excess blank sHDL (FIG. 1C).Moreover, the preliminary data showed that the cholesterol modifiedPCSK9 siRNA (PCSK9 Cho-siRNA) could be quantitatively incorporated intosHDL. Although free PCSK9 Cho-siRNA can knockdown PCSK9 in HepG2 cellsdue to the increased uptake of siRNA induced by cholesterol conjugation,PCSK9 siRNA-sHDL is still better able to knockdown PCSK9 protein inHepG2 cells in vitro (FIG. 1D-F).

EXAMPLE III

This example demonstrates that co-localized delivery of antigen andadjuvant by sHDL leads to potent immune response. FIG. 4A presents aschematic of antigens and adjuvants-loaded sHDL. When a MHC class Iantigen peptide (CD8+ T cell epitope peptide SIINFEKL derived fromovalbumin) was incubated with functional lipids-containing sHDL, theantigen peptide was quantitatively conjugated to functional lipids ofsHDL, as can be seen by the disappearance of functional lipids andappearance of lipid-peptide conjugates (FIG. 2B). The cholesterolmodified CpG (Cho-CpG) was also shown to be quantitatively incorporatedinto sHDL (FIG. 2C). After 1 primary dose and two booster doses, theantigen and CpG-loaded sHDL (sHDL-Ag/CpG) elicited more potent immuneresponses than the mixture of antigens and CpG in Montanide(CpG+Montanide is one of the most potent experimental adjuvant currentlyundergoing clinical evaluations) (FIG. 2D).

FIG. 3 shows a schematic of the synthesis ofsHDL-CSSSIINFEK(FITC)L/CpG(SEQ ID NO:386).

FIG. 4 shows homogenous particle size of sHDL-Ag/CpG as analyzed bycryoEM and dynamic light scattering.

FIGS. 5A and 5B show that compared with free antigen form, antigendelivery via sHDL significantly prolongs antigen presentation bydendritic cells.

FIG. 6 shows that sHDL-Ag/CpG significantly enhances elicitation ofantigen-specific CD8+ T cells, compared with vaccination with freeantigen mixed with conventional adjuvants.

FIG. 7 shows sHDL-Ag/CpG vaccination elicits strong CD8+ T cellresponses in tumor-bearing mice and reduces tumor growth.

EXAMPLE IV

This example demonstrates that sHDL delivering alpha-galactosylceramide,a glycolipid ligand for CD1-d to activate induction of natural killer Tcells.

FIG. 8 shows that compared with free soluble form, alpha-GalCerdelivered via sHDL significantly enhanced CD1d presentation ofantigen-presenting cells.

FIG. 9 shows that lyophilization offers a convenient method oflarge-scale synthesis of sHDL loaded with alpha-GalCer.

EXAMPLE V

This example demonstrates that preformed high densitylipoprotein-mimicking nanodiscs can be readily coupled with antigen (Ag)peptides and adjuvants, producing stable, ultrasmall nanoparticles thatmarkedly improve Ag/adjuvant co-delivery to lymphoid organs and achievesustained Ag presentation on dendritic cells.

Lipids and peptides conducive to nanodisc formation were firstidentified. DMPC lipid films were hydrated and added with a series ofApoA1-mimetic peptides, followed by thermal cycling between 50° C. and4° C. A subset of peptides, including 22A and D-amino acids of 22A, wereidentified that produced clear sHDL suspensions, stable for one monthwhen stored at 4° C. (FIG. 13a ). In addition, use of phospholipids withtransition temperature (Tm) below RT (e.g. POPC and DOPC with Tm=−2° C.and −17° C., respectively) produced murky liposomal suspension, whereaslipids with high Tm (e.g. DPPC and DMPC with Tm=41° C. and 24° C.,respectively) formed clear sHDL suspensions in the presence of 22A (FIG.13b ), showing flexibility in the materials design. Based on their size,homogeneity, and long-term stability, 22A and DMPC as the key componentsof nanodisc vaccines were chosen for further investigation.

To achieve intracellular release of Ag within APCs viareduction-sensitive conjugation of Ag on sHDL, we synthesizeddioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP, FIG. 14) and incorporated PDP into sHDL (4 mol %).When incubated for 30 min at RT with Ag peptides modified with acysteine-serine-serine (CSS) linker (see, e.g., Hirosue, S., et al.,Vaccine 28, 7897-7906 (2010)), sHDL nanodiscs were efficientlysurface-decorated with various Ag peptides (e.g., OVA₂₅₇₋₂₆₄, a modelCD8α+ T-cell epitope Ag from ovalbumin; gp100₂₅₋₃₃, melanoma-associatedAg; and Adgpk, neo-antigen in MC-38), and subsequent incubation withCho-CpG for 30 min at RT led to almost complete (˜98%) insertion of CpGinto sHDL, producing nanodiscs co-loaded with Ag and CpG (termedsHDL-Ag/CpG, with ˜6.5 Ag peptides and ˜1 CpG molecule per nanodisc,FIG. 15; Table 4). sHDL-Ag/CpG exhibited uniform disc-like morphologywith an average diameter of 10.5±0.5 nm and polydispersity index of0.20±0.02 (FIGS. 16a and 16b ). Importantly, sHDL-Ag/CpG could bereadily sterile-filtered and stored frozen at −20° C. for at least 8weeks before thawing at 37° C., without negatively affecting itshomogeneity (FIG. 16c ).

TABLE 4 % of PDP- % of lipid Cho-CpG converted Inserted SizeFormulations to Ag-lipid into sHDL (d · nm) PDI sHDL- 92.0 ± 3.5% 98.5 ±1.1% 10.5 ± 0.5 0.20 ± 0.02 CSSSIINFEKL/ CpG sHDL-gp100/CpG 91.6 ± 2.7%98.2 ± 1.5% 10.3 ± 0.5 0.23 ± 0.03 sHDL-Adpgk/CpG 91.1 ± 3.1% 96.5 ±1.8% 10.8 ± 0.3 0.22 ± 0.02

The impact of nanodiscs on Ag presentation was next examined. Bonemarrow derived dendritic cells (BMDCs) pulsed for 24 h withsHDL-CSSSIINFEKL/CpG (SEQ ID NO:384) presented OVA₂₅₇₋₂₆₄ SIINFEKL witha greater efficiency than BMDCs treated with free Ag peptides admixedwith CpG or sHDL-CSSSIINFEKL (SEQ ID NO:384), as determined by stainingDCs with the 25-D1.16 mAb directed against SIINFEKL-H-2K^(b) complexes(FIGS. 16 d; 17 a and 17 b). Interestingly, DCs pulsed with freeSIINFEKL+CpG efficiently presented Ag for the first 6 h of incubation,but Ag presentation decreased precipitously past 6 h (FIGS. 16e and 16f; FIG. 17c ), suggesting initial direct Ag binding to MHC-I molecules,followed by rapid Ag degradation or disassociation. In contrast, Agpresentation with sHDL-Ag/CpG gradually increased over time, achieving˜9-fold greater levels at 24 h and maintaining ˜4-fold higher levelseven at 48 h, compared with free SIINFEKL+CpG.

Intrigued by prolonged Ag presentation, the process of nanodisc uptakeand Ag localization using CSS-SIINFEK_((FITC))L was investigated;SIINFEKL modified with FITC at ε-amino group in the lysine residue isknown to retain its binding capacity to H-2K^(b) molecules (see, e.g.,Saini, S. K. et al. Proc. Natl. Acad. Sci. U. S. A. 110, 15383-15388(2013). JAWSII cells (immortalized immature DCs) incubated with freeAg(FITC)+CpG displayed weak fluorescence signal on the plasma membraneat 6 h, and only dim fluorescence was observed by 24 h (FIG. 16 g; FIG.18). In stark contrast, sHDL-Ag(FITC)/CpG treatment led to strong FITCsignal co-localized with endosomes/lysosomes by 6 h, and robust Ag(FITC)signal was detected on cell membranes by 24 h and sustained up to 48 h.In addition, nanodiscs containing Rh-PE or Texas Red-labeled-22A werepredominantly found within endosomes/lysosomes, indicating cellularuptake of intact whole nanodiscs (FIG. 19). To assess the impact ofprolonged Ag presentation on T-cell cross-priming, BMDCs were treatedwith free Ag peptides+CpG or sHDL-Ag/CpG for 24 or 48 h, and then addedSIINFEKL-specific, H-2K^(b)-restricted B3Z T-cell hybridomas. BMDCspulsed with sHDL-Ag/CpG promoted strong B3Z T-cell activation even after48 h incubation, whereas free Ag peptides+CpG induced minimal B3Z T-cellactivation beyond the 24 h period (FIG. 16h ). Moreover, sHDL-Ag/CpGpotently stimulated DC maturation (FIG. 20). Altogether, whereas free Agpeptide was rapidly loaded and dissociated from MHC-I molecules on cellmembranes, nanodiscs facilitated intracellular delivery of Ag/CpG andmediated their sustained release within endosomes/lysosomes, therebypromoting durable Ag presentation, APC maturation, and cross-primingCD8α+ T-cells in vitro.

The impact of nanodiscs on lymphatic delivery of Ag/CpG and induction ofCTL responses in vivo (see, e.g., Reddy, S. T. et al. Nat. Biotechnol.25, 1159-1164 (2007)) was next investigated. C57BL/6 mice injectedsubcutaneously at tail base with 31 nmol free CSS-SIINFEK_((FITC))L hadminimal FITC signal in inguinal dLNs after 1 day (see, e.g., FIG. 21a ),potentially due to systemic dissemination of small MW Ag peptide ordirect Ag binding on non-APCs at the injection site (see, e.g., Melief,C. J. & van der Burg, S. H. Nat. Rev. Cancer 8, 351-360 (2008). Incontrast, sHDL-Ag group exhibited markedly increased FITC signal in dLNs(p<0.01, FIG. 21a ), with Ag(FITC) and Cy5-tagged 22A co-localizedwithin dLNs (FIG. 22). Similarly, injection of 2.3 nmol Cy5-taggedCho-CpG in sHDL increased its LN accumulation, compared with injectionin free soluble form (p<0.01, FIG. 21b ). These results showed that sHDLnanodisc promoted co-delivery of Ag and CpG to dLNs. C57BL/6 mice werenext immunized with 15.5 nmol Ag and 2.3 nmol CpG (non-fluorophoretagged), and peripheral blood mononuclear cells (PBMCs) were analyzedfor the frequency of SIINFEKL-MHC-I tetramer+ CD8α+ T-cells. The mixtureof free Ag peptides (SIINFEKL or CSS-SIINFEKL) and CpG induced 1-3%Ag-specific CTLs after the third immunization (FIGS. 21c and 21d ). Asthe benchmark, animals with the equivalent doses of Ag and CpGemulsified in water-in-oil Montanide were also vaccinated (see, e.g.,Speiser, D. E. et al. J. Clin. Invest. 115, 739-746 (2005); Fourcade, J.et al. J. Immunother. 31, 781-791 (2008)). Ag+CpG+Montanide elicited ˜2%Ag-specific CTLs after priming; however, no further T-cell expansion wasobserved even after the third immunization, consistent with a recentstudy reporting dysfunction and deletion of high-avidity T-cells afterrepeated immunizations with a depot-forming water-in-oil adjuvant (see,e.g., Rezvani, K. et al. Haematologica 96, 432-440 (2011); Hailemichael,Y. et al. Nat. Med. 19, 465-472 (2013)). In striking contrast,sHDL-Ag/CpG group elicited a peak frequency of ˜21% Ag-specific CD8α+T-cells after the third vaccination (29-fold greater than solubleSIINFEKL+CpG and 9-fold greater than Ag+CpG+Montanide, p<0.0001, FIGS.21c and 21d ). When challenged with 2×10⁵ B16OVA cells, mice immunizedwith sHDL-Ag/CpG had no detectable tumor masses up to 28 days, with 40%of animals surviving for more than 200 days, whereas mice immunized withfree Ag peptides+CpG or Ag+CpG+Montanide all succumbed to tumors withmarginal survival benefits (FIGS. 21e and 2f ). Importantly, throughoutsuch experiments, no signs of toxicity or autoimmunity in animalsimmunized multiple times with sHDL-Ag/CpG were observed.

Experiments were conducted to rule out the possibility that CSS-modifiedpeptides or Cho-CpG dissociated from sHDL-Ag/CpG in vivo wereresponsible for the strong CTL responses. Introducing the CSS linker toSIINFEKL and replacing free CpG with Cho-CpG in free soluble formresulted in minimal T-cell responses, and the physical mixture of Ag,CpG, and sHDL also elicited weak CTL responses (FIG. 21g ). In contrast,sHDL-Ag/CpG nanodiscs drastically improved CTL responses, elicitingremarkable 41-fold greater frequency of Ag-specific CD8α+ T-cells thanCSSSINFEKL+Cho-CpG group (day 35,p<0.0001, FIG. 21g ), with CTLsprimarily exhibiting CD44^(high)CD62L^(low) effector phenotype androbust IFN-γ⁺ ELISPOT responses (FIG. 21h ; FIG. 23).

The anti-tumor efficacy of sHDL in tumor-bearing mice was evaluated.Therapeutic sHDL vaccination in mice bearing B16OVA melanoma led tostrong Ag-specific CTL responses with significantly slowed tumor growthand extended animal survival (FIG. 24). Nanodisc vaccines were nexttested using non-immunogenic B16F10 melanoma as a more clinicallyrelevant model. After confirming incorporation of gp100₂₅₋₃₃ togetherwith Cho-CpG into nanodiscs (FIG. 15; Table 4), mice were treated with15.5 nmol Ag and 2.3 nmol CpG on days 4 and 11 post subcutaneousinoculation of B16F10 cells. Vaccinations with sHDL-gp100/CpG elicitedrobust CTL responses, generating 22-fold higher frequency ofgp100-specific CTLs than free gp100+CpG (day 17,p<0.0001, FIG. 25a ;FIG. 26), leading to significantly delayed tumor growth and prolongedanimal survival, compared with the free gp100+CpG group that had noeffects (FIGS. 25b and 25c ).

Finally, to demonstrate the utility of the platform technology forvaccination against neo-antigens, the murine MC-38 colon carcinoma modelrecently reported to harbor a single-epitope mutation within Adpgkprotein (ASMTNRELM→ASMTNMELM (SEQ ID NO:383)) was employed, with theneo-epitope presented in MHC-I H-2D^(b) molecules (see, e.g., Yadav, M.et al. Nature 515, 572-576 (2014)). The Adpgk neo-antigen mutation inMC-38 cells was confirmed by cDNA sequencing (FIG. 25d ; FIG. 27) andsynthesized sHDL-Adpgk/CpG by mixing nanodiscs with the neo-epitopemodified with the CSS-linker and Cho-CpG. C57BL/6 mice were inoculatedsubcutaneously with 10⁵ MC-38 cells and treated with 15.5 nmol Adpgkmutated peptide and 2.3 nmol CpG. Mice treated with free Adpgk Ag+CpGhad similar levels of Adpgk-specific CD8α+ T-cells as non-immunized,MC-38-bearing mice, whereas sHDL-Adpgk/CpG markedly enhanced CTLresponses (day 23, p<0.001, FIG. 25e ). In addition, sHDL-Adpgk/CpGinduced polyfunctional IFN-γ⁺ and IFN-γ⁺TNF-α⁺ Adpgk-specific CD8α+T-cells (2.5-fold and 7-fold greater than the free Adpgk+CpG group,p<0.05 and p<0.001, respectively, FIG. 25f ). Importantly, therapeutictreatments with sHDL-Adpgk/CpG substantially slowed MC-38 tumor growthand extended animal survival, in contrast to the traditional solubleAdpgk+CpG vaccine with no statistically significant effects on tumorgrowth or survival (median survival: 54 d versus 33 d, respectively,p<0.01, FIG. 25g and FIG. 25h ).

EXAMPLE VI

This example pertains to the materials and methods for Example V.

Materials

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and rhodamine(Rhod)-labeled DOPE (DOPE-Rhod) were purchased from Avanti Polar Lipids(Alabaster, Ala.). ApoA1 mimetic peptide (22A), OVA₂₅₇₋₂₆₄ SIINFEKL,CSSSIINFEKL (SEQ ID NO:384), CSSSIINFEK(FITC)L(SEQ ID NO:386),hgp100₂₅₋₃₃ KVPRNQDWL, CSSSKVPRNQDWL, and Adpgk mutant peptide ASMTNMELM(SEQ ID NO:383) were synthesized by GenScript Corp. (Piscataway, N.J.).CSSASMENMELM was synthesized by AnaSpec (Fremont, Calif.). Theoligodeoxynucleotide TLR9 ligand CpG 1826(5′-tccatgacgttcctgacgtt-3′(SEQ ID NO:382), lower case letters representphosphorothioate backbone), CpG 1826 modified with cholesterol at the 3′end (Cho-CpG), and Cy5 modified Cho-CpG were synthesized by IntegratedDNA Technologies (Coralville, Iowa). HPLC grade methanol andacetonitrile were purchased from Fisher Scientific (Pittsburgh, Pa.).Fetal bovine serum (FBS), penicillin-streptomycin, β-mercaptoethanol andACK lysis buffer were purchased from Life Technologies (Grand Island,N.Y.). Granulocyte macrophage colony stimulating factor (GM-CSF) wasfrom GenScript Corp. (Piscataway, N.J.). Anti-mouse CD16/32, CD86-PE,CD40-APC, CD62L-PECy7, and 25-D1.16 mAb-PE against SIINFEKL/H-2K^(b)were from eBioscience (San Diego, Calif.). Anti-mouse CD8α-APC,CD44-FITC, TNF-α-FITC, IFN-γ-PE, and CD11c-PECy7 were from BD Bioscience(San Jose, Calif.). Tetramer H-2K^(b)-SIINFEKL-PE and TetramerH-2D^(b)-KVPRNQDWL-PE was purchased from Beckman Coulter (Brea, Calif.).Tetramer/H-2D^(b)-ASMTNMELM-PE (SEQ ID NO:383) was kindly provided bythe NIH Tetramer Core Facility (Atlanta, Ga.). We obtained B3Z CD8α+ Tcell hybridoma from Dr. N. Shastri (University of California, Berkeley);B16OVA from Dr. Kenneth Rock (University of Massachusetts, Amherst,Mass.); and MC-38 cells from Dr. Weiping Zou (University of Michigan,Ann Arbor, Mich.).

Methods Synthesis and Characterization of DOPE-PDP

Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP) was synthesized as reported previously withslight modifications (see, e.g., Kuai, R., et al. Mol. Pharm. 7,1816-1826 (2010)). Briefly, DOPE, SPDP (succinimidyl 3-(2-pyridyldithio)propionate) and triethylamine (1:1:1.5 molar ratio) were dissolved inchloroform. The mixture was reacted in the dark for 5 h. The reactionprogress was monitored by thin layer chromatography (TLC), using thefollowing mixture as the developing solvent:chloroform/methanol/water=65/25/4 (volume ratio). After TLC indicateddisappearance of the starting materials and appearance of afaster-running spot, the reaction mixture was dried by rotaryevaporation and purified on a silica gel column.

Synthesis of sHDL Co-Loaded with Antigen Peptides and CpG

DMPC and DOPE-PDP (molar ratio=96:4) were dissolved in chloroform. Themixture was dried with nitrogen flow and place under vacuum for at least1 h. The resulting lipid film was hydrated in 10 mM sodium phosphatebuffer (0.3117 g/L NaH₂PO₄.H₂O and 2.0747 g/L Na₂HPO₄.7H₂O, pH 7.4) andsonicated in a bath sonicator for 10 min, followed by probe sonicationfor another 2.5 min. ApoA1 mimetic peptide 22A dissolved in endotoxinfree water was added to the above mixture (22A:lipids=1:7.5 molarratio), which was then subjected to heating (50° C.) for 3 min andcooling (ice water) for 3 min, with 3 cycles in total, to obtain sHDL.

To conjugate tumor antigen peptides to sHDL, cysteine terminated tumorantigen peptides dissolved in endotoxin free water were added to theabove sHDL (antigen peptides:DOPE-PDP=2.5:1, molar ratio) and incubatedat room temperature with gentle shaking on an orbital shaker. Unreactedtumor antigen peptides were removed by using Zeba Spin Desalting columns(Pierce) following the manufacturer's instructions. The conjugationefficiency of tumor antigen peptides was calculated based on thedecrease of absorbance signal associated with DOPE-PDP as determined byHPLC. Briefly, 200 μl sHDL formulations were freeze-dried andreconstituted in 300 μl methanol. The mixture was filtered by a 0.22 μmPTFE filter and analyzed with a Shimadzu HPLC system using a Vydac 219TPDiphenyl column (4.6 mm×250 mm ID). The two solvents used for the HPLCanalysis consisted of water:trifluoroacetic acid=100:0.5 (mobile phaseA) and methanol: acetonitrile:trifluoroacetic acid=50:50:0.05 (mobilephase B) (0-75 min, 15-100%). The flow rate was 0.4 mL/min and thedetection wavelength was 220 nm. The loading efficiency of tumor antigenpeptides in sHDL was confirmed by using FITC-labeled peptides andmeasuring the fluorescence intensity of sHDL formulations at Ex=490 nmand Em=520 nm after dissolving the formulations in PBS containing 1%Triton X-100.

To load CpG in sHDL, different concentrations (0-200 μg/mL) ofcholesterol modified CpG (Cho-CpG) were incubated with sHDL at roomtemperature with gentle shaking on an orbital shaker. The amount of CpGincorporated into sHDL and free CpG was analyzed by gel permeationchromatography (GPC). Briefly, the sHDL formulations were diluted in PBSto a concentration of 0.5 mg/mL 22A peptide. The formulations werefiltered through a 0.22 μm filter and analyzed with a Shimadzu HPLCsystem equipped with a TSKgel G2000SWx1 column (7.8 mm ID×30 cm, TosohBioscience LLC). The flow rate of mobile phase PBS (pH 7.4) was set at0.7 mL/min, and the detection wavelength was set at 260 nm for CpG.

Characterization of Peptide/CpG-Loaded sHDL Formulations

The sHDL formulations were diluted to 0.5 mg/mL 22A with PBS, and theparticle sizes were measured by dynamic light scattering (DLS, ZetasizerNano ZSP, Malvern, UK). The morphology of sHDL was observed bytransmission electron microscopy (TEM) after proper dilution of theoriginal samples. Briefly, 3 μL of the sample solution was deposited ona carbon film-coated 400 mesh copper grid (Electron Microscopy Sciences)and dried for 1 minute. The samples were then negatively-stained with 5droplets of 1% uranyl acetate solution, excessive solutions on the gridwere blotted, and the grid was dried before TEM observation. All imageswere acquired on JEM 1200EX electron microscope (JEOL USA, Peabody,Mass.) equipped with an AMT XR-60 digital camera (Advanced MicroscopyTechniques Corp. Woburn, Mass.).

Preparation of BMDCs

BMDCs were prepared as described previously (see, e.g., Lutz, M. B., etal. J. Immunol. Methods 223, 77-92 (1999)). Briefly, femur and tibiawere harvested aseptically from C57BL/6 mice, and the bone marrow wasflushed into a petri dish using a 5 mL syringe (26 G needle) loaded withBMDC culture media (RPMI 1640 supplemented with 10% FBS, 100 U/mLpenicillin, 100 μg/ml streptomycin, 50 μM β-mercaptoethanol, and 20ng/ml GM-CSF). Cells were collected by passing the cell suspensionthrough a cell strainer (mesh size=40 μm), followed by centrifugation.Cells were seeded into non-tissue culture treated petri-dish at adensity of 2×10⁵ cells/ml, cultured at 37° C. with 5% CO₂. Culture mediawere refreshed on days 3, 6, 8, and 10, and BMDCs were used for thefollowing assays on days 8-12.

Activation of BMDCs

Immature BMDCs were plated at 1×10⁶ cells/well in 12-well plates. After24 h, BMDCs were washed once with PBS and incubated with 75 nM of CpG indifferent formulations or 0.5 μg/mL LPS (positive control) for 24 h at37° C. with 5% CO₂. BMDCs were harvested, washed with FACS buffer (1%BSA in PBS), incubated with anti-CD16/32 at room temperature for atleast 10 min, and then stained with fluorophore-labeled antibodiesagainst CD11c, CD40, CD80, and CD86 at room temperature for 30 min.Finally, cells were washed twice by FACS buffer, resuspended in 2 μg/mlDAPI solution, and analyzed by flow cytometry (Cyan 5, Beckman Coulter,USA).

Antigen Presentation on BMDCs

Immature BMDCs were plated at 1×10⁶ cells/well in 12-well plates 24 hprior to use. BMDCs were washed with PBS and incubated with 75 nM CpGand/or 500 nM antigen peptide in various formulations in complete mediafor different lengths of time (2, 6, 24, and 48 h). BMDCs were thenharvested, washed with FACS buffer, incubated with anti-CD16/32 at roomtemperature for at least 10 min, and stained with PE-conjugatedanti-mouse SIINFEKL/H-2K^(b) mAb 25-D1.16 at room temperature for 30min. Cells were then washed, resuspended in 2 μg/ml DAPI solution, andanalyzed by flow cytometry (Cyan 5, Beckman Coulter, USA).

Confocal Microscopy Imaging of the Intracellular Trafficking of sHDL

JAWSII cells (ATCC, Manassas, Va.) were seeded at 1×10⁶ cells on 35 mmpetri dishes (MatTek Corp., Ashland, Mass.) that have beenpre-equilibrated with the complete cell culture media and culturedovernight. To investigate the intracellular delivery profiles of antigenpeptides, JAWSII cells were incubated with the physical mixture of freeCSSSIINFEK(FITC)L (SEQ ID NO:386) and CpG, or sHDL-CSSSIINFEK(FITC)L/CpGfor different lengths of time (6, 24, and 48 h). Cells were then washed3 times with PBS and incubated for 30 min at 37° C. with 50 nMLysoTracker® Red DND-99 (Invitrogen) and 2 μg/mL Hoechst inphenol/serum-free media to stain lysosomes and nuclei, respectively. Inparallel, to study the intracellular delivery profiles of structuralcomponents of sHDL, the lipid layers of sHDL were incorporated withDOPE-Rhod by adding 0.5 mol % DOPE-Rhod in the initial lipid film, while22A peptide of sHDL was labeled by incubating pre-formed sHDL with TexasRed®-X succinimidyl ester (Life Technologies) and passing TexasRed-labeled sHDL through a desalting column to remove the unreacted dye.The resulting fluorophore-tagged sHDL formulations were incubated withJAWSII cells at 37° C. with 5% CO₂. After 24 h incubation, cells werewashed 3 times with PBS and then incubated for 30 min at 37° C. with 500nM LysoTracker® Green DND-26 (Invitrogen) and 2 μg/mL Hoechst inphenol/serum-free media to stain lysosomes and nuclei, respectively.JAWSII cells were then imaged using a confocal microscope (Nikon A1).

Activation of B3Z CD8+ T Hybridoma Cells with sHDL

BMDCs were plated at 5×10⁴ cells/well in a U-bottom 96-well plate. Afterovernight culture, BMDCs were washed with PBS and incubated withdifferent formulations of SIINFEKL (20, 100 and 500 nM) and CpG (3, 15,and 75 nM) for 24 h or 48 h at 37° C. Cells were then carefully washed 3times with PBS, and 10⁵ B3Z CD8+ T hybridoma cells/well were added inRPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 55 μMβ-mercaptoethanol, 1 mM pyruvate and 100 U/mL penicillin and 100 μg/mLstreptomycin. After 24 hr of incubation, cells were pelleted viacentrifugation (1500 rcf, 7 min), the media were carefully aspirated,and 150 μL CPRG/lysis buffer (0.15 mM chlorophenolred-β-D-galactopyranoside (CPRG), 0.1% Triton-X 100, 9 mM MgCl2, 100 μMmercaptoethanol in PBS) was added. The plates were incubated at 37° C.in the dark for 90 min, after which the absorbance of releasedchlorophenol red was measured at 570 nm using a microplate reader.

In Vivo Immunization Studies

Animals were cared for following federal, state, and local guidelines.All work performed on animals was in accordance with and approved byUniversity Committee on Use and Care of Animals (UCUCA) at University ofMichigan, Ann Arbor. Female C57BL/6 mice of age 6-8 weeks (HarlanLaboratories) were immunized with different formulations containingantigen peptides (15.5 nmol/mouse) and CpG (2.3 nmol/mouse) in 100 μlvolume by subcutaneous injection at the tail base on indicated timepoints. In some studies, antigen peptide and CpG emulsified in Montanideserved as a positive control (see, e.g., Speiser, D. E., et al. J. Clin.Invest. 115, 739-746 (2005); Fourcade, J., et al. J. Immunother. 31,781-791 (2008); Karbach, J., et al. Int. J. Cancer 126, 909-918 (2010)).Briefly, antigen peptide (155 nmol) and CpG (23 nmol) in 0.5 mL PBS werethoroughly emulsified in 0.5 mL Montanide until the mixture washomogeneous.

For lymph node draining studies, C57BL/6 mice were injected with freeCSSSIINFEK(FITC)L(SEQ ID NO:386), sHDL-CSSSIINFEK(FITC)L, freeCho-CpG(Cy5), or sHDL-Cho-CpG(Cy5). After 24 h, inguinal lymph nodeswere harvested, and FITC or Cy5 fluorescence signal was measured withIVIS optical imaging system (Caliper Life Sciences).

For prophylactic tumor challenge studies, vaccinated animals werechallenged on day 8 after last immunization by subcutaneous injection of2×10⁵B16OVA cells/mouse on the right flank. Tumor growth was monitoredevery other day, and the tumor volume throughout this study wascalculated by the following equation (see, e.g., Gorrin-Rivas, M. J., etal. Clin. Cancer Res. 6, 1647-1654 (2000)): tumorvolume=length×width²×0.52. Animals were euthanized when the tumor massesreached 1.5 cm in diameter or when animals became moribund with severeweight loss or ulceration.

For therapeutic tumor vaccination studies, C57BL/6 mice were inoculatedwith tumor cells (2×10⁵ B16OVA cells, 2×10⁵B16F10 cells, or 1×10⁵ MC38cells per mouse) on the right flank by subcutaneous injection on day 0.For B16OVA and B16F10 studies, mice were vaccinated on days 4 and 11with different formulations containing 15.5 nmol of tumor antigenpeptides (SIINFEKL and hgp100, respectively) and 2.3 nmol of CpG. ForMC-38 studies, mice were vaccinated on days 10, 17, and 24 with 15.5nmol of ASMTNMELM (SEQ ID NO:383) and 2.3 nmol of CpG in either sHDL orfree soluble form. Tumor growth was monitored as indicated above.

Peptide-MHC Tetramer Assay

Immunized mice were analyzed for the percentages of tumorantigen-specific CD8α+ T cells among peripheral blood mononuclear cells(PBMCs) using the tetramer staining assay, as described previously (see,e.g., Ochyl, L. J. & Moon, J. J. J. Vis. Exp. e52771 (2015)). In brief,100 μl of blood was drawn from each mouse on indicated time points bysubmandibular bleeding, and red blood cells were lysed with ACK lysisbuffer. PBMCs were then washed with FACS buffer and blocked byanti-CD16/32 antibody and incubated with peptide-MHC tetramer taggedwith PE (e.g. H-2K^(b)-restricted SIINFEKL, H-2D^(b)-restrictedKVPRNQDWL, or H-2D^(b)-restricted ASMTNMELM (SEQ ID NO:383)) for 30 minat room temperature. Samples were then incubated with anti-CD8α-APC for20 min on ice. Cells were washed twice with FACS buffer and resuspendedin 2 μg/ml DAPI solution for analysis by flow cytometry (Cyan 5, BeckmanCoulter, USA).

ELISPOT and Intracellular Cytokine Staining Assays

For ELISPOT assay, spleens from immunized mice were harvestedaseptically, processed into single cell suspensions for each mouse, andseeded at 3×10⁵ splenocytes per well in 96-well PVDF plates (EMDMillipore) pre-incubated overnight with IFN-γ coating Ab (R&D Systems).Splenocytes were co-incubated with antigen peptides (2.5 μg/ml) orcontrols for 24 hours. Assays were completed using sequentialincubations with biotinylated-secondary Ab, streptavidin-alkalinephosphatase (Sigma Chemical), and NBT/BCIP substrate (Surmodics).Developed spots were enumerated using an AID iSpot Reader (AutoimmunDiagnostika GmbH, Germany). For intracellular cytokine staining (ICS)assay, 100-150 μL peripheral blood collected from vaccinated mice waslysed with ACK lysis buffer, washed with PBS, and were plated at ˜10million cells/mL in 50 μL T cell media (RPMI 1640 supplemented with 10%FBS, 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1 mM pyruvate and 100U/mL penicillin and 100 μg/mL streptomycin, HEPES, and non-essentialamino acids) in 96-well U bottom plates. Cells were pulsed with 10 μg/mLantigen peptides for 6 hours with protein transport inhibitor, brefeldinA (BD Biosciences), added during the last 4 h of incubation. Cells werethen washed twice with ice-cold FACS buffer (1% BSA in PBS), followed byincubation with anti-CD16/32 for at least 10 minutes and anti-CD8α for20 min on ice. Cells were then fix/permeabilized for 20 min on ice andthen stained with anti-IFN-γ-PE and anti-TNF-α-FITC for 30 min on ice.After extensive washing, cells were analyzed by flow cytometry.

cDNA Sequencing of Neo-Epitope (Adpgk) in MC-38 Cells

Total RNA was extracted from MC-38 cells by the RNeasy® mini Kit(QIAGEN) following the manufacturer's instructions. The first-strandcDNA was synthesized using 1 μg of total RNA with the SuperScript™ IIIFirst-Strand Synthesis SuperMix Kit (Invitrogen). Adpgk cDNA withlengths of 250 bp and 485 bp were selectively amplified by using thefollowing two sets of sequence specific primers. Primer 1:TGCCAACCGCTTCATCTTCT (forward primer) and GGTAGACCAGCGTGTGGAAA (reverseprimer); Primer 2: CTCCAACGGGGCCATGAATA (forward primer) andCGTGGGAAAGACCTGCTGAT (reverse primer). The amplification was performedusing the SuperScript One Step RT-PCR System (Invitrogen). The finalcDNA products were visualized in 1.5% agarose gels with ethidiumbromide, and the Adpgk cDNA bands were cut and purified using thePureLink® Quick Gel Extraction and PCR Purification Combo Kit(Invitrogen). The purified cDNA was sequenced by the Sanger sequencingmethod (see, e.g., Sanger, F., Nicklen, S. & Coulson, A. R. DNAsequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S. A. 74, 5463-5467 (1977)) at the University of Michigan DNA SequencingCore.

EXAMPLE VII

This example describes neo-antigen vaccination using othernanoparticles, including sHDL, liposomes, and gold nanoparticles (FIG.29), and the generation of multivalent neo-antigen vaccination usingmultiple neo-antigen peptides (FIG. 28).

Preparation of sHDL Loaded with Multivalent Neo-Antigens

To prepare nanodisc-based multivalent peptide vaccine, multipleneo-antigen peptides (M30 and M27) modified with CSS linker atN-terminus were conjugated to DOPE-PDP in dimethylformamide at roomtemperature for 3 hours, followed by dilution with 10× water andlyophilization to obtain lipid-peptide conjugates. The conjugate wasmixed with DMPC and 22A in acetic acid and lyophilized. The resultingpowder was then subjected to heating (50° C.) for 3 min and cooling (icewater) for 3 min, with 3 cycles in total, to obtain sHDL loaded withdifferent neo-antigens (sHDL-M30/M27). Alternatively, the conjugate wasdissolved in DMSO and incubated with preformed sHDL to obtain sHDLloaded with different neo-antigens (sHDL-M30/M27). Any unincorporatedneo-antigen peptides were removed by passing through a desalting column.The loading efficiency was analyzed by HPLC. Cholesterol-CpG wasincubated with the above sHDL at room temperature for 30 min to obtainthe nanodisc-based multivalent peptide vaccine (sHDL-M30/M27/CpG).

Preparation of Liposomes Loaded with Neo-Antigens

To prepare liposome-based neo-antigen vaccines, DMPC and DOPE-PDP (molarratio=92:8) were dissolved in chloroform. The mixture was dried withnitrogen flow and placed under vacuum for at least 1 h. The resultinglipid film was hydrated in 10 mM sodium phosphate buffer (0.3117 g/LNaH2PO4.H2O and 2.0747 g/L Na2HPO4.7H2O, pH 7.4) and sonicated in a bathsonicator for 10 min, followed by probe sonication for another 2.5 minto obtain liposomes. The neo-antigen peptide Adpgk was conjugated toliposomes after incubation of CSS-modified Adpgk peptides withPDP-displaying liposomes, followed by desalting column-based separationof unconjugated peptides. The conjugation efficiency was analyzed byHPLC. Cholesterol-CpG was incubated with the above liposomes at roomtemperature for 30 min to obtain the liposome-based neo-antigen peptidevaccine (lip-Adpgk/CpG).

Preparation of Spiky Gold Nanoparticle-Based Neo-Antigen Peptide Vaccine

To obtain spiky gold nanoparticles (AuNPs), citrate gold nanoparticleswere first prepared by boiling HAuCl₄ aqueous solution with sodiumcitrate. They were sequentially added with HAuCl₄, HCl, AgNO₃, andascorbic acid at room temperature under vigorous stirring to form AuNPsvia seed-mediated growth method. As-synthesized AuNPs were purified andconcentrated by centrifugation with 0.01% SDS. AuNP-based peptidevaccine was prepared by thiol-mediated surface decoration of neo-antigenpeptides on AuNPs followed by polylC and CpG layer loading throughelectrostatic complexation. Briefly, peptide vaccine wassurface-conjugated to AuNPs by overnight incubation of AuNPs withCSS-modified neo-antigen peptide, CSS-ASMTNMELM (SEQ ID NO:383). Anyunreacted peptide was removed from AuNP conjugates by centrifugation. Toload polylC and CpG via electrostatic interaction, polyethylene glycol(average Mn 6,000)-modified polyethyleneimine (branched, average Mw˜25,000) (PEG-PEI) was employed. The peptide-conjugated AuNPs were mixedwith PEG-PEI for 10 min, purified from excessive PEG-PEG bycentrifugation, and added to polyIC and CpG mixture solution in 10 mMNaCl. After 5 min, the mixture was transferred to PEG-PEI solution in 10mM NaCl, and the salt concentration was step-wise increased to 150 mMNaCl by the increment of 50 mM every 5 min. Finally, the crude mixturesolution was centrifuged with 0.01% tween20 to remove any unbound polylCand CpG.

Intracellular Cytokine Staining

C57BL/6 mice were vaccinated with nanodisc-based multivalent neo-antigenpeptide vaccine (sHDL-M30/M27/CpG) on day 0, 7, and 14. Seven days afterthe last vaccination, 100-150 μL peripheral blood collected fromvaccinated mice was lysed with ACK lysis buffer, washed with PBS, andwere plated at ˜10 million cells/mL in 50 μL T cell media (RPMI 1640supplemented with 10% FBS, 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1mM pyruvate and 100 U/mL penicillin and 100 μg/mL streptomycin, HEPES,and non-essential amino acids) in 96-well U bottom plates. Cells wereco-cultured with 50000 BMDCs/well and pulsed with 20 μg/mL of M30 or M27peptide for 6 hours with protein transport inhibitor, brefeldin A (BDBiosciences), added during the last 4 h of incubation. Cells were thenwashed twice with ice-cold FACS buffer (1% BSA in PBS), followed byincubation with anti-CD16/32 for at least 10 minutes and anti-CD8α andanti-CD4 for 20 min on ice. Cells were then fix/permeabilized for 20 minon ice and then stained with anti-IFN-γ-PE for 30 min on ice. Afterextensive washing, cells were analyzed by flow cytometry. The resultsshown in FIG. 28 indicate that sHDL-M30/M27/CpG generated highfrequencies of CD4+ T-cells against neo-antigen M30 (FIG. 28A) and CD8+T-cells against neo-antigen M27 (FIG. 28B).

Therapeutic Study

For therapeutic tumor vaccination studies, C57BL/6 mice were inoculatedwith tumor cells (1×10⁵ MC38 cells per mouse) on the right flank bysubcutaneous injection on day 0. Mice were vaccinated on days 10 and 17with 15.5 nmol of ASMTNMELM (SEQ ID NO:383) and 2.3 nmol of CpG (or 15μg polylC/mouse) formulated in either liposomes or soluble forms. Forthe group of mice immunized with AuNPs, intratumoral administration ofAuNPs modified with Adpgk and adjuvants was performed on days 10 (bothw/ and w/o laser groups) and 16 (only w/o laser group) with 12 nmol ofASMTNMELM (SEQ ID NO:383), 5.2 nmol of CpG, and 83 μg polylC per mouse.Laser was directly irradiated to tumor tissues at 1.2 W/cm² for 5 minusing 808 nm CW diode laser.

On indicated time points, PBMCs were collected and stained forAdpgk-specific CD8+ T cells among PBMCs via tetramer staining, followedby cytometric analysis. The tetramer staining of PBMCs indicated thatAdpgk-containing liposomes and AuNPs all generated strongerneo-antigen-specific CD8+ T cell responses, compared with vaccinationwith soluble peptide plus adjuvants (FIG. 29A). In addition, tumorgrowth was monitored every other day, and the tumor volume throughoutthis study was calculated by the following equation: tumorvolume=length×width²×0.52. Animals were euthanized when the tumor massesreached 1.5 cm in diameter or when animals became moribund with severeweight loss or ulceration. The results indicated that Adpgk-containingnanoparticles, including liposomes and AuNPs, slowed tumor progression,compared with vaccination with soluble peptide and CpG (FIG. 29B).

EXAMPLE VIII

This example describes HDL preparation using full length protein.

Heat/Cool (Lipid Film) Method

First, lipid films were prepared by weighing out and dissolving thedesired amount of lipid in chloroform, followed by solvent evaporationunder a stream of nitrogen gas. Excess chloroform was subsequentlyremoved by placing vials in a vacuum oven set to room temperatureovernight. The following day, the lipid films were hydrated with warmphosphate buffer, followed by vortexing and sonication by both bath andprobe sonication. Once translucent, ApoA-I protein solution was warmedand added to the lipid suspension. The resulting protein-lipid mixturewas vortexed and thermal-cycled between 50° C. and room temperature (10minutes each) for 3 cycles. The solution turned clear by the end of thethird cycle, indicating the formation of HDL. The HDL suspension wasfiltered using 0.22 um syringe filters and characterized for purityusing gel permeation chromatography (GPC) and size by dynamic lightscattering (DLS).

Both DMPC and SM lipids were able to form HDL with ApoA-I using thismethod. GPC showed ApoA-I-DMPC and ApoA-I-SM HDL were 93.5% and 94.4%pure, respectively, with small amounts of un-complexed lipid (˜5.59 min)and ApoA-I (˜8.7 min) impurities (see, FIG. 35).

Neither POPC, DMPG:DPPG-NH₄ ⁺, nor EggPC were able to form HDL withApoA-I via this method (FIG. 36). Suspensions remained cloudy even afterrepeated thermal cycling, and DLS measurements show that the averageparticle sizes were >1 um in diameter with high polydispersity.

DLS data confirmed the sizes of ApoA-I-DMPC and ApoA-I-SM HDL to be ˜8.5nm in diameter (FIG. 36).

Cholate/BioBeads SM—Second Method

For this method, lipid films of DMPG:DPPG-NH₄ ⁺(4:1 mol/mol), POPC, orEggPC were prepared using the method described above. After excesssolvent removal under vacuum, lipid films were hydrated with warm ApoA-Isolution and vortexed, resulting in a cloudy white suspension. Sodiumdeoxycholate, dissolved to 20 mg/mL in PBS, was slowly titrated into thecloudy protein-lipid suspension. The mixture was vortexed and incubatedat 37° C. until the suspension turned clear. Cholate was then removed byincubation with BioBeads SM-2 (˜20 mg BioBeads per 1 mL solution) at 37°C. with gentle agitation for 3 hours. The resulting HDL suspension wastransferred to a new vial and filtered using 0.22 um syringe filters andcharacterized for purity using gel permeation chromatography (GPC) andsize by dynamic light scattering (DLS).

EggPC, POPC, and DMPG:DPPG-NH₄ ⁺ lipids were able to form HDL withApoA-I using this method. GPC analysis showed that particles were onlyabout 85% pure (˜7.7 min), however, with excess cholate appearing ˜11.6minutes and very small amounts of uncomplexed lipid (˜5.5 min) andprotein (˜9 min) present (see, FIG. 37A).

Neither increasing the incubation time of HDL with BioBeads norincreasing the amount of BioBeads added resulted in additional cholateremoval, but rather started to disrupt the HDL particles themselves (asshown in the last slide, splitting of HDL peak with no decrease incholate peak) (see, FIG. 37B).

EXAMPLE IX

This example describes HDL preparation using apolipoprotein mimetics.

In contrast to heterogenous HDL formation by recombinant ApoA-I,homogeneous sHDL nanodiscs using ApoA-I-mimetic peptide and variouslipids were produced by the following two methods.

Co-Lyophilization

Appropriate amount of lipids and ApoA-I mimetic peptide were dissolvedin glacial acetic acid, which was removed by freeze-drying overnight.Phosphate-buffered saline (PBS, pH=7.4) was added to the freeze-driedpowder, which was cycled 3 times between 50° C. (3 min) and 20° C. (3min) with gentle shaking to obtain sHDL. The obtained sHDL nanoparticleswere sterilized by passing through 0.22 um syringe filters andcharacterized for purity using gel permeation chromatography (GPC) andsize by dynamic light scattering (DLS).

Thin Film or Mixing and Thermal Cycling

Appropriate amount of lipids were dissolved in chloroform, which wasremoved by putting under nitrogen flow and then in vacuum oven to form athin lipid film. The lipid film was hydrated with 10 mM sodium phosphatebuffer (pH 7.4) using bath sonication. ApoA-I mimetic peptide was addedto the lipid dispersion, which was cycled 3 times between 50° C. (3 min)and 20° C. (3 min) with gentle shaking to obtain sHDL nanoparticles. Theobtained sHDL nanoparticles were sterilized by passing through 0.22 umsyringe filters and characterized for purity using gel permeationchromatography (GPC) and size by dynamic light scattering (DLS).

A broad range of phospholipids with different chain lengths andtransition temperatures, including POPC, DLPC, DMPC, SM, DPPC, HSPC, andDSPC were all able to efficiently complex with ApoA-1 mimetic peptide(22A) to form homogeneous sHDL nanoparticles using either of the abovetwo methods. GPC showed the purity of formed sHDL nanoparticles was over99%, with little to no free peptide or uncomplexed lipids (see, FIG.38). DLS further confirmed the good homogeneity of HDL nanoparticles.Depending on the lipid composition, the sizes of sHDL nanoparticles werebetween 8.3-10.8 nm, which were all close to that of endogenous HDL(see, FIG. 39).

EXAMPLE X

This example describes the lack of immunogenicity or autoimmunity aftermultiple immunizations with “blank” sHDL nanodiscs (without antigen oradjuvant) thereby indicating safety of the ApoA-I-mimetic peptide-basednanodiscs.

Mice immunized multiple times with sHDL composed of phospholipids andApoA-I-mimetic peptide 22A did not display any signs of toxicity,autoimmunity, nor immune responses directed against the ApoA1-mimeticpeptide 22A. The results indicate that “blank” sHDL nanodiscs are notimmunogenic on their own and they don't elicit any detectable autoimmuneresponses.

Measurement of antibody titers against 22A peptide: ELISA plates werecoated with 22A peptide in PBS (1 μg/mL) with 100 μL/well and incubatedovernight at 4° C. Plates were blocked with 1% BSA in PBS for 2 h, and100 μL of 4-fold serial dilutions of serum was added to each 96-well andincubated for 1 hour at room temperature. Wells were incubated withrabbit anti-mouse IgG-HRP (1:5000 dilution) for 1 h at room temperature,followed by addition of the HRP substrate, TMB. The enzymatic reactionwas stopped by adding 2N H₂SO₄, and the absorbance at 450 nm (OD450) wasmeasured using a microplate reader. The highest dilution with twice theabsorbance of background was considered as the end-point dilution titer.

Additional experiments were conducted wherein C57BL/6 mice wereimmunized with sHDL-CpG (equivalent to 2.3 nmol CpG per dose) for 3times in an 1-week interval. FIG. 40 shows the percent of 22A-specificCD4+ T cells (a), 22A-specific CD8+ T cells (b) among PBMCs one weekafter the third vaccination, and (c) the titers of IgG antibody against22A one week after the third vaccination. Data represent mean±SD from arepresentative experiment (n=3) from 2 independent experiments. NS,non-statistically significant. Importantly, throughout such experiments,signs of toxicity, autoimmunity, immune responses directed against theApoA1-mimetic peptide 22A in animals immunized multiple times withsHDL-Ag/CpG were not observed (see, FIG. 40).

EXAMPLE XI

This example shows in vivo data where nanodisc vaccination is combinedwith immuno stimulatory agent (e.g., immune checkpoint inhibitors). Inaddition, this example demonstrates therapeutic efficacy of combinationimmunotherapy between nanodisc vaccination and immune checkpointblockade. In addition, this example demonstrates the use of multipletumor antigens delivered by “cocktail” of nanodiscs with or withoutimmune checkpoint blockade.

Preparation of a Single or Cocktail of Tumor Antigens on sHDL Nanodiscs

To conjugate tumor antigen peptides to sHDL, cysteine terminated tumorantigen peptides dissolved in endotoxin free water were added to theabove sHDL (antigen peptide:DOPE-PDP=2.5:1, molar ratio) and incubatedat room temperature with gentle shaking on an orbital shaker. Toconstruct sHDL nanodiscs with multi-antigens, each antigen peptide wasreacted with DOPE-PDP (antigen peptide:DOPE-PDP=1.5:1, molar ratio) for1 h in dimethylformamide (DMF), which was removed by freeze-drying afterdilution with endotoxin-free water. The lipid-peptide conjugates wereadded to pre-formed sHDL and incubated for 30 min at room temperature.Unreacted tumor antigen peptides were removed by using Zeba SpinDesalting columns (Pierce) following the manufacturer's instructions.The conjugation efficiency of tumor antigen peptides was calculatedbased on the decrease of absorbance signal associated with DOPE-PDP asdetermined by HPLC.

Combination Immunotherapy between sHDL Nanodisc Vaccination and ImmuneCheckpoint Blockade

For therapeutic tumor vaccination studies with MC-38 cells, C57BL/6 micewere inoculated with 1×10⁵ MC38 cells per mouse on the right flank bysubcutaneous injection on day 0 and vaccinated on days 10, 17, and 24with 15.5 nmol of ASMTNMELM (SEQ ID NO:383) and 2.3 nmol of CpG ineither sHDL or free soluble form. For the combinatorial immunotherapyagainst MC-38 tumor, mice were inoculated subcutaneously with 1×10⁵ MC38cells on day 0 and vaccinated on days 10, and 17 with 15.5 nmol ofASMTNMELM (SEQ ID NO:383)and 2.3 nmol of CpG in either sHDL or freesoluble form. Anti-mouse PD-1 (100 μg/mouse) was administeredintraperitoneally on days 1 and 4 after each vaccination. For B16F10studies, mice were inoculated subcutaneously with 1×10⁵ B16F10 cells onday 0 and vaccinated on days 4, 11, and 18 with indicated formulations(10 nmol of each antigen peptide and 2.3 nmol of CpG). For thecombinatorial immunotherapy against B16F10 tumor, anti-mouse PD-1 andanti-mouse CTLA-4 (each 100 μg/mouse) antibodies were administeredintraperitoneally on days 1 and 4 after each vaccination. Tumor growthwas monitored.

MC-38 Treatment with Neo-Antigen sHDL Vaccination Combined withAnti-PD-1 Antibody Therapy

Therapeutic vaccination with sHDL-Adpgk/CpG induced polyfunctionalIFN-γ⁺ and IFN-γ⁺TNF-α⁺ Adpgk-specific CD8α+ T-cells and substantiallyslowed MC-38 tumor growth (FIG. 41A), compared with the traditionalsoluble Adpgk+CpG vaccine. However, no tumor rejection was observed ineither vaccine groups, potentially due to immunosuppression within tumormicroenvironment, as we detected high expression levels of programmedcell death-1 (PD-1) and its ligand PD-L1 among tumor-infiltrating CD8α+T-cells and tumor cells, respectively. In order to block theimmunosuppressive PD-1/PD-L1 pathway, experiments combined the vaccineswith anti-PD-1 antibodies (αPD-1). Combination immunotherapy withsHDL-Adpgk/CpG and αPD-1 treatment generated robust neoantigen-specificCTL responses and led to complete tumor regression in ˜88% mice (FIG.41B), compared with ˜25% rate of tumor regression in the solubleAdpgk+CpG+αPD-1 group.

B16F10 Treatment with a Cocktail of Neo-Antigens on sHDL NanodiscsCombined with Dual Anti-PD-1/CTLA-4 Antibody Therapy

Experiments were conducted to evaluate the nanodisc platform in amelanoma model with B16F10 cells, as they are highly aggressive, poorlyimmunogenic, and hence hard to treat with conventional cancer vaccines.To prevent tumor immune escape by loss of a single mutant allele,experiments sought to elicit broad-spectrum T-cell responses byemploying multiple antigens (multiAgs), including recently reportedB16F10 mutated neo-epitopes (MHC I-restricted M27 and MHC II-restrictedM30) as well as MHC I-restricted epitope from tyrosinase-related protein2 (TRP2, a melanoma-associated Ag), all loaded in the same nanodiscs.C57BL/6 mice inoculated subcutaneously with 10⁵ B16F10 cells werevaccinated with sHDL-multiAgs/CpG, eliciting a total of ˜30%Ag-specific, IFN-γ⁺ CD8α+ and CD4+ T-cells in peripheral blood, comparedwith only 1-3% induced by the soluble multiAgs+CpG ormultiAgs+CpG+Montanide groups (p<0.0001, FIG. 42A). Vaccination withsHDL-multiAgs/CpG significantly inhibited B16F10 tumor growth, comparedwith the soluble or Montanide vaccines (p<0.0001, FIG. 42B). Notably,removing either M27/M30 or TRP2 from sHDL-multiAgs/CpG compromised itstherapeutic efficacy, suggesting the benefits of broad CTL responsesagainst neo-antigens and tumor-associated antigens (FIG. 42C). Weevaluated sHDL-multiAgs/CpG combined with dual immune checkpointinhibitors. Combination immunotherapy with sHDL-multiAgs/CpG andαPD-1/αCTLA-4 treatment led to an impressive rate of B16F10 tumorrejection with ˜90% of mice free of tumor, whereas the solublemultiAgs+CpG+αPD-1/αCTLA-4 treatment mediated tumor regression in ˜38%of animals (FIG. 42D).

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-247. (canceled)
 248. A composition comprising a nanoparticle, whereina biomacromolecule agent is associated with (e.g., complexed,conjugated, encapsulated, absorbed, adsorbed, admixed) the nanoparticle.249. The composition of claim 248, wherein the nanoparticle is selectedfrom the group consisting of sHDL nanoparticles, fullerenes, endohedralmetallofullerenes buckyballs, trimetallic nitride templated endohedralmetallofullerenes, single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nanotubes, gold nanorods, silver nanorods,single-walled and multi-walled boron/nitrate nanotubes, carbon nanotubepeapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, any nanostructures, microstructures, or theirderivatives formed using layer-by-layer processes, self-assemblyprocesses, or polyelectrolytes, microparticles, quantum dots,superparamagnetic nanoparticles, nanorods, cellulose nanoparticles,glass and polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold nanoparticles, silver nanoparticles, carbonnanoparticles, iron nanoparticles, and modified micelles.
 250. Thecomposition of claim 248, wherein the biomacromolecule is an antigen.251. The composition of claim 250, wherein the antigen is conjugated tothe outer surface of the nanoparticle or the adjuvant is conjugated tothe outer surface of the nanoparticle.
 252. The composition of claim250, wherein the antigen is selected from the group consisting ofalpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27,cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusionprotein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11,hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I,OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphateisomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1,Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, andTRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2,MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGS),SCP-1, Hom/Mel-40, PRAIVIE, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL,H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MA-9, CA 72-4, CAM17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7,telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1,CO-029, GE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1,PSA, TAG-72-4, CA 19FGF-5, G250, Ga733 (EpCAM), human EGFR protein orits fragments, such as human EGFR residues 306-325 (SCVRACGADSYEMFEDGVRK(SEQ ID NO:374)) and residues 897-915 (VWSYGVTVWELMTFGSKPY (SEQ IDNO:375)), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1,SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associatedprotein), TAAL6, TAG72, TLP, TPS, WT1 (and WT1-derived peptidesequences: WT1 126-134 (RMFP NAPYL (SEQ ID NO:376)), WT1 122-140(SGQARMFPNAPYLPSCLES (SEQ ID NO:377)), and WT1 122-144(SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO:378)), MUC1 (and MUC1-derivedpeptides and glycopeptides such as RPAPGS (SEQ ID NO:379), PPAHGVT (SEQID NO:380), and PDTRP (SEQ ID NO:381))), LMP2, EGFRvIII, Idiotype, GD2,Ras mutant, p53 mutant, Proteinase3 (PR1), Survivin, hTERT, Sarcomatranslocation breakpoints, EphA2, EphA4, LMW-PTP, PAP, ML-IAP, AFP, ERG(TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgen receptor, CyclinB1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin,sLe(animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5,SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK,HMWMAA, AKAP-4, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1,FAP, PDGFR-alpha, PDGFR-β, MAD-CT-2, Fos-related antigen 1, ERBB2,Folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K, fms-related tyro-sinekinase 1 (FLT1, best known as VEGFR1), KDR, PADRE, TA-CIN (recombinantHPV16 L2E7E6), SOX2 and aldehyde dehydrogenase.
 253. The composition ofclaim 250, wherein the antigen is associated with (e.g., complexed,conjugated, encapsulated, absorbed, adsorbed, admixed) a hydrophobicmolecule, wherein the hydrophobic molecule is a lipid molecule, whereinthe lipid molecule is a membrane-forming lipid molecule or anon-membrane-forming lipid molecule.
 254. The composition of claim 253,wherein the lipid molecule is selected from the group consisting ofphospholipids such as lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1l-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, sterols such as cholesterol andderivatives thereof such as cholestanol, cholestanone, cholestenone,coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, nonphosphorous containing lipids suchas, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and mixtures thereof; or wherein thelipid molecule is selected from the group consisting of fatty acids andderivatives or analogs thereof including oleic acid, lauric acid, capricacid (n-decanoic acid), myristic acid, palmitic acid, stearic acid,linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.); or whereinthe lipid molecule is selected from the group consisting of a lipidmolecule modified with PEG (PEG-lipid), wherein the lipid moleculemodified with PEG is selected from the group consisting of PEG coupledto dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG),PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE),PEG conjugated to ceramides, PEG conjugated to cholesterol or aderivative thereof, PEG-C-DOMG, 2 KPEG-DMG, and mixtures thereof. 255.The composition of claim 248, wherein the composition further comprisesan adjuvant associated with (e.g., complexed, conjugated, encapsulated,absorbed, adsorbed, admixed) with the nanoparticle, wherein the adjuvantis encapsulated within the sHDL nanoparticle, wherein the adjuvant isselected from the group consisting of CPG, polylC, poly-ICLC, 1018 ISS,aluminum salts (for example, aluminum hydroxide, aluminum phosphate),Amplivax, BCG, CP-870,893, CpG7909, CyaA, dSLIM, Cytokines (such asGM-CSF, IL-2, IFN-a, Flt-3L), IC30, IC31, Imiquimod, ImuFact IMP321, ISPatch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipidA, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system,PLGA microparticles, imiquimod, resiquimod, gardiquimod, 3M-052, SRL172,Virosomes and other Virus-like particles, YF-17D, VEGF trap,beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA),3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands(such as C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide,α-mannosylceramide, α-fructosylceramide, β-galatosylceramide,β-mannosylceramide), STING agonists (e.g. cyclic dinucleotides,including Cyclic [G(3′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(3′,5′)p],Cyclic [G(2′,5′)pA(2′,5′)p], Cyclic diadenylate monophosphate, Cyclicdiguanylate monophosphate), CL401, CL413, CL429, Flagellin, RC529,E6020, imidazoquinoline-based small molecule TLR-7/8a (including itslipidated analogues), virosomes, AS01, AS02, AS03, AS04, AS15, IC31,CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), bacterialtoxins (such as CT, and LT), any derivative of an adjuvant, and anycombination of adjuvant.
 256. The composition of claim 248, wherein thenanoparticle is a sHDL nanoparticle, wherein the sHDL nanoparticlecomprises a mixture of at least one phospholipid and at least one HDLapolipoprotein or apolipoprotein mimetic, wherein the HDL apolipoproteinis selected from the group consisting of apolipoprotein A-I (apo A-I),apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4),apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E), wherein thephospholipid is selected from the group consisting ofdipalmitoylphosphatidylcholine (DPPC),dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol,1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],phosphatidylcholine, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, and combinations thereof.
 257. The compositionof claim 256, wherein the HDL apolipoprotein mimetic is an ApoA-Imimetic, wherein the thiol-reactive phospholipid isdioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), wherein the ApoA-I mimetic is described by anyof SEQ ID NOs: 1-336 and (SEQ ID NO: 341) WDRVKDLATVYVDVLKDSGRDYVSQF,(SEQ ID NO: 342) LKLLDNWDSVTSTFSKLREOL, (SEQ ID NO: 343)PVTOEFWDNLEKETEGLROEMS, (SEQ ID NO: 344) KDLEEVKAKVQ, (SEQ ID NO: 345)KDLEEVKAKVO, (SEQ ID NO: 346) PYLDDFQKKWQEEMELYRQKVE, (SEQ ID NO: 347)PLRAELQEGARQKLHELOEKLS, (SEQ ID NO: 348) PLGEEMRDRARAHVDALRTHLA,(SEQ ID NO: 349) PYSDELRQRLAARLEALKENGG, (SEQ ID NO: 350)ARLAEYHAKATEHLSTLSEKAK, (SEQ ID NO: 351) PALEDLROGLL, (SEQ ID NO: 352)PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 353) PVLESFVSFLSALEEYTKKLN,(SEQ ID NO: 352) PVLESFKVSFLSALEEYTKKLN, (SEQ ID NO: 354)TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 355) QTVTDYGKDLME, (SEQ ID NO: 356)KVKSPELOAEAKSYFEKSKE, (SEQ ID NO: 357) VLTLALVAVAGARAEVSADOVATV,(SEQ ID NO: 358) NNAKEAVEHLOKSELTOOLNAL, (SEQ ID NO: 359)LPVLVWLSIVLEGPAPAOGTPDVSS, (SEQ ID NO: 360) LPVLVVVLSIVLEGPAPAQGTPDVSS,(SEQ ID NO: 361) ALDKLKEFGNTLEDKARELIS, (SEQ ID NO: 362)VVALLALLASARASEAEDASLL, (SEQ ID NO: 363) HLRKLRKRLLRDADDLQKRLAVYOA,(SEQ ID NO: 364) AQAWGERLRARMEEMGSRTRDR, (SEQ ID NO: 365)LDEVKEQVAEVRAKLEEQAQ, (SEQ ID NO: 236) DWLKAFYDKVAEKLKEAF,(SEQ ID NO: 366) DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA, (SEQ ID NO: 367)PVLDLFRELLNELLEALKQKL, (SEQ ID NO: 368) PVLDLFRELLNELLEALKQKLA,(SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK, (SEQ ID NO: 369)PVLDLFRELLNELLEALKQKLA, (SEQ ID NO: 370) PVLDLFRELLNELLEALKKLLK,(SEQ ID NO: 371) PVLDLFRELLNELLEALKKLLA, (SEQ ID NO: 372)PLLDLFRELLNELLEALKKLLA, and (SEQ ID NO: 373) EVRSKLEEWFAAFREFAEEFLARLKS.


258. The composition of claim 248, wherein the average particle size ofthe nanoparticle is between 6 to 500 nm.
 259. The composition of claim248, wherein the biomacromolecule agent is a peptide selected from thegroup consisting of an Adrenocorticotropic Hormone (ACTH), a growthhormone peptide, a Melanocyte Stimulating Hormone (MSH), Oxytocin,Vasopressin, Corticotropin Releasing Factor (CRF), a CRF-relatedpeptide, a Gonadotropin Releasing Hormone Associated Peptide (GAP),Growth Hormone Releasing Factor (GRF), Lutenizing Hormone ReleaseHormone (LH-RH), an orexin, a Prolactin Releasing Peptide (PRP), asomatostatin, Thyrotropin Releasing Hormone (THR), a THR analog,Calcitonin (CT), a CT-precursor peptide, a Calcitonin Gene RelatedPeptide (CGRP), a Parathyroid Hormone (PTH), a Parathyroid HormoneRelated Protein (PTHrP), Amylin, Glucagon, Insulin, an Insulin-likepeptide, NeuroPeptide Y (NPY), a Pancreatic Polypeptide (PP), Peptide YY(PYY), Cholecystokinin (CCK), a CCK-related peptide, Gastrin ReleasingPeptide (GRP), Gastrin, a Gastrin-related peptide, a Gastrin inhibitorypeptide, Motilin, Secretin, Vasoactive Intestinal Peptide (VIP), aVIP-related peptide, an Atrial-Natriuretic Peptide (ANP), a BrainNatriuretic Peptide (BNP), a C-Type Natriuretic Peptide(CNP), atachykinin, an angiotensin, a renin substrate, a renin inhibitor, anendothelin, an endothelin-related peptide, an opioid peptide, a thymicpeptide, an adrenomedullin peptide, an allostatin peptide, an amyloidbeta-protein fragment, an antimicrobial peptide, an antioxidant peptide,an apoptosis related peptide, a Bag Cell Peptide (BCPs), Bombesin, abone Gla protein peptide, a Cocaine and Amphetamine Related Transcript(CART) peptide, a cell adhesion peptide, a chemotactic peptide, acomplement inhibitor, a cortistatin peptide, a fibronectin fragment, afibrin related peptide, FMRF, a FMRF amide-related peptide (FaRP),Galanin, a Galanin-related peptide, a growth factor, a growthfactor-related peptide, a G-Therapeutic Peptide-Binding Proteinfragment, Gualylin, Uroguanylin, an Inhibin peptide, Interleukin (IL),an Interleukin Receptor protein, a laminin fragment, a leptin fragmentpeptide, a leucokinin, Pituitary Adenylate Cyclase ActivatingPolypeptide (PAPCAP), Pancreastatin, a polypeptide repetitive chain, asignal transducing reagent, a thrombin inhibitor, a toxin, a trypsininhibitor, a virus-related peptide, an adjuvant peptide analog, AlphaMating Factor, Antiarrhythmic Peptide, Anorexigenic Peptide, Alpha-1Antitrypsin, Bovine Pineal Antireproductive Peptide, Bursin, C3 PeptideP16, Cadherin Peptide, Chromogranin A Fragment, ContraceptiveTetrapeptide, Conantokin G, Conantokin T, Crustacean CardioactivePeptide, C-Telopeptide, Cytochrome b588 Peptide, Decorsin, DeliciousPeptide, Delta-Sleep-Inducing Peptide, Diazempam-Binding InhibitorFragment, Nitric Oxide Synthase Blocking Peptide, OVA Peptide, PlateletCalpain Inhibitor (P1), Plasminogen Activator Inhibitor 1, Rigin,Schizophrenia Related Peptide, Sodium Potassium Atherapeutic PeptidaseInhibitor-1, Speract, Sperm Activating Peptide, Systemin, a Thrombinreceptor agonist, Tuftsin, Adipokinetic Hormone, Uremic Pentapeptide,Antifreeze Polypeptide, Tumor Necrosis Factor (TNF), Leech [DesAsp10]Decorsin, L-Ornithyltaurine Hydrochloride, P-AminophenylacetylTuftsin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro,Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly,D-Ala-Leu, D-D-D-D-D, D-D-D-D-D-D, N-P-N-A-N-P-N-A, V-A-I-T-V-L-V-K,V-G-V-R-V-R, V-I-H-S, V-P-D-P-R, Val-Thr-Cys-Gly, R-S-R, Sea UrchinSperm Activating Peptide, a SHU-9119 antagonist, a MC3-R antagonist, aMC4-R antagonist, Glaspimod, HP-228, Alpha 2-Plasmin Inhibitor, APCTumor Suppressor, Early Pregnancy Factor, Gamma Interferon, GlandularKallikrei N-1, Placental Ribonuclease Inhibitor, Sarcolecin BindingProtein, Surfactant Protein D, Wilms' Tumor Suppressor, GABAB 1bReceptor Peptide, Prion Related Peptide (iPRP13), Choline BindingProtein Fragment, Telomerase Inhibitor, Cardiostatin Peptide, EndostatinDerived Peptide, Prion Inhibiting Peptide, N-Methyl D-Aspartate ReceptorAntagonist, and C-PeptideAnalog; or 177Lu-DOTA0-Tyr3-Octreotate,Abarelix acetate, ADH-1, Afamelanotidec, melanotan-1, CUV1647,Albiglutide, Aprotinin, Argipressin, Atosiban acetate, Bacitracin,Bentiromide, a BH3 domain, Bivalirudin, Bivalirudin trifluoroacetatehydrate, Blisibimod, Bortezomib, Buserelin, Buserelin acetate,Calcitonin, Carbetocin, Carbetocin acetate, Cecropin A and B,Ceruletide, Ceruletide diethylamine, Cetrorelix, Cetrorelix acetate,Ciclosporine, Cilengitidec, EMD121974, Corticorelin acetate injection,hCRF, Corticorelin ovine triflutate, corticorelin trifluoroacetate,Corticotropin, Cosyntropin, ACTH 1-24, tetracosactide hexaacetate,Dalbavancin, Daptomycin, Degarelix acetate, Depreotide trifluoroacetate(plus sodium pertechnetate), Desmopressin acetate, Desmopressin DDAVP,Dulaglutide, Ecallantide, Edotreotide (plus yttrium-90), Elcatoninacetate, Enalapril maleate (or 2-butanedioate), Enfuvirtide,Eptifibatide, Exenatide, Ganirelix acetate, Glatiramer acetate,Glutathion, Gonadorelin, Gonadorelin acetate, GnRH, LHRH, Goserelin,Goserelin acetate, Gramicidin, Histrelin acetate, Human calcitonin,Icatibant, Icatibant acetate, IM862, oglufanide disodium, KLAKLAK,Lanreotide acetate, Lepirudin, Leuprolide, Leuprolide acetate,leuprorelin, Liraglutide, Lisinopril, Lixisenatide, Lypressin,Magainin2, MALP-2Sc, macrophage-activating lipopeptide-2 synthetic,Nafarelin acetate, Nesiritide, NGR-hTNF, Octreotide acetate,Oritavancin, Oxytocin, Pasireotide, Peginesatide, Pentagastrin,Pentetreotide (plus indium-111), Phenypressin, Pleurocidin, Pramlintide,Protirelin, thyroliberin, TRH, TRF, Salmon calcitonin, Saralasinacetate, Secretin (human), Secretin (porcine), Semaglutide, Seractideacetate, ACTH, corticotropin, Sermorelin acetate, GRF 1-29, Sinapultide,KL4 in lucinactant, Sincalide, Somatorelin acetate, GHRH, GHRF, GRF,Somatostatin acetate, Spaglumat magnesium (or sodium) salt, Substance P,Taltirelin hydrate, Teduglutide, Teicoplanin, Telavancin, Teriparatide,Terlipressin acetate, Tetracosactide, Thymalfasin, thymosin a-1,Thymopentin, Trebananib, Triptorelin, Triptorelin pamoate,Tyroserleutide, Ularitide, Vancomycin, Vapreotide acetate, Vasoactiveintestinal peptide acetate, Vx-001c, TERT572Y, Ziconotide acetate, α5-α6Bax peptide, and β-defensin.
 260. The composition of claim 248, whereinthe biomacromolecule agent is a nucleic acid molecule, wherein thenucleic acid molecule is selected from RNA, siRNA, microRNA,interference RNA, mRNA, replicon mRNA, RNA-analogues, and DNA.
 261. Thecomposition of claim 248, wherein the composition is used as a vaccineagainst a pathogen, wherein the pathogen is selected from erythropoieticporphyries, T2 diabetes, antifibrinolytic, central diabetes insipidus,delaying the birth in case of threat of premature birth, antibiotic,cystic fibrosis, angina, anticoagulant in patients with unstable anginaundergoing PTCA or PCI, systemic lupus erythematosus, hypercalcemia,osteoporosis, pagets disease, carbetocin works as an oxytocic,antihemorrhagic and uterotonic drug in the peripheral nervous system,prevention of uterine atony, induction, and control postpartum bleedingor hemorrhage, stimulant of the gastric secretion, for treathormone-sensitive cancers of the prostate and breast, inhibition ofpremature LH surges in women undergoing controlled ovarian stimulation,immunosuppression in organ transplantation to prevent rejection,peritumoral brain edema, diagnosis of ACTHdependent Cushing's syndrome,allergies, ankylosing spondylitis, psoriasis, chorioditis, erythema,keratitis, sclerosis, dermatomyositis, rheumatoid arthritis,Stevens-Johnson Syndrome, ulcerative colitis, diagnosis ofadrenocortical insufficiency, antibiotic, systemic infections caused bygram positive organisms, nocturnal enuresis, nocturia, and stoppage ofbleeding or hemorrhage in haemophilia A patients, acute hereditaryangioderma, postmenopausal osteoporosis, anti-parathyroid, Paget'sdisease, hypercalcaemia, hypertension, AIDS/HIV-1 infection, acutecoronary syndrome, unstable angina undergoing PCI, Alzheimer's andParkinson's disease, inhibition of premature LH surges in womenundergoing controlled ovarian hyperstimulation, Relapsing-RemittingMultiple Sclerosis, hepatic insufficiency, wound healing, inflammationof respiratory tract, asthenia, release of follicle-stimulating hormone(FSH) and luteinizing hormone (LH) from the anterior pituitary,stimulate the secretion of gonadotropin during disturbances fertility,and diagnosis of the functional capacity and response of thegonadotropes of the anterior pituitary, for skin lesions, surface woundsand eye infections, postmenopausal osteoporosis, Paget's disease,hypercalcaemia, hereditary angioedema, immune system related diseases,acromegaly, anticoagulant, fibroids and endometriosis, central diabetesinsipidus, Cushing's syndrome, diabetic foot ulcers, treatment ofcentral precocious puberty, uterine fibriods and endometriosis,vasodilatory, natriuretic, diuretic and neurohormonal effects,acromegaly, carcinoid syndrome, acute bacterial skin and skin structureinfections, initiation or improvement of uterine contractions, andcontrol postpartum bleeding or hemorrhage, hematide Chronic kidneydisease associated anemia, stomatitis, pharyngitis, diagnosticassessment of thyroid function, postmenopausal osteoporosis,hypercalcaemia, diagnosis of pancreatic exocrine dysfunction, andgastrinoma, Zollinger-Ellison syndrome, prevention of RDS in prematureinfants, and meconium aspiration syndrome, acute variceal bleeding,allergic rhinitis and conjunctivitis, spinocerebellardegeneration/ataxia, Short Bowel Syndrome, antibiotic, bactericidal,teriparatide is the only anabolic (i.e., bone growing) agent indicatedfor use in postmenopausal women with osteoporosis, Cortrosyn Analogue ofadrenocorticotrophic hormone (ACTH) used for diagnostic purposes,treatment of adrenal insufficiency, epilepsia, Chronic hepatitis B,chronic hepatitis C, primary and secondary immune deficiencies, acutedecompensated heart failure, colitis, esophageal variceal bleeding inpatients with cirrhotic liver disease and AIDS-related diarrhea,sarcoidosis and acute lung injury, and severe chronic pain.
 262. Thecomposition of claim 261, wherein the composition is used within methodsfor cancer immunotherapy.
 263. A method of treating a condition,disorder and/or disease in a subject, comprising administering acomposition as described in claim 248 to the subject.
 264. The method ofclaim 263, wherein the condition, disorder and/or disease is peripheralischemia, cancer, an inflammatory disorder, and/or a genetic disorder.265. The method of claim 263, wherein the biomacromolecule is a peptide,wherein the peptide and condition, disorder and/or disease is selectedfrom the group consisting of each row in Table 1; or wherein thebiomacromolecule is a nucleic acid, wherein the nucleic acid molecule isselected from RNA, siRNA, microRNA, interference RNA, mRNA, repliconmRNA, RNA-analogues, and DNA.
 266. The method of claim 263, wherein thecondition, disorder and/or disease is selected from erythropoieticporphyries, T2 diabetes, antifibrinolytic, central diabetes insipidus,delaying the birth in case of threat of premature birth, antibiotic,cystic fibrosis, angina, anticoagulant in patients with unstable anginaundergoing PTCA or PCI, systemic lupus erythematosus, hypercalcemia,osteoporosis, pagets disease, carbetocin works as an oxytocic,antihemorrhagic and uterotonic drug in the peripheral nervous system,prevention of uterine atony, induction, and control postpartum bleedingor hemorrhage, stimulant of the gastric secretion, for treathormone-sensitive cancers of the prostate and breast, inhibition ofpremature LH surges in women undergoing controlled ovarian stimulation,immunosuppression in organ transplantation to prevent rejection,peritumoral brain edema, diagnosis of ACTHdependent Cushing's syndrome,allergies, ankylosing spondylitis, psoriasis, chorioditis, erythema,keratitis, sclerosis, dermatomyositis, rheumatoid arthritis,Stevens-Johnson Syndrome, ulcerative colitis, diagnosis ofadrenocortical insufficiency, antibiotic, systemic infections caused bygram positive organisms, nocturnal enuresis, nocturia, and stoppage ofbleeding or hemorrhage in haemophilia A patients, acute hereditaryangioderma, postmenopausal osteoporosis, anti-parathyroid, Paget'sdisease, hypercalcaemia, hypertension, AIDS/HIV-1 infection, acutecoronary syndrome, unstable angina undergoing PCI, Alzheimer's andParkinson's disease, inhibition of premature LH surges in womenundergoing controlled ovarian hyperstimulation, Relapsing-RemittingMultiple Sclerosis, hepatic insufficiency, wound healing, inflammationof respiratory tract, asthenia, release of follicle-stimulating hormone(FSH) and luteinizing hormone (LH) from the anterior pituitary,stimulate the secretion of gonadotropin during disturbances fertility,and diagnosis of the functional capacity and response of thegonadotropes of the anterior pituitary, for skin lesions, surface woundsand eye infections, postmenopausal osteoporosis, Paget's disease,hypercalcaemia, hereditary angioedema, immune system related diseases,acromegaly, anticoagulant, fibroids and endometriosis, central diabetesinsipidus, Cushing's syndrome, diabetic foot ulcers, treatment ofcentral precocious puberty, uterine fibriods and endometriosis,vasodilatory, natriuretic, diuretic and neurohormonal effects,acromegaly, carcinoid syndrome, acute bacterial skin and skin structureinfections, initiation or improvement of uterine contractions, andcontrol postpartum bleeding or hemorrhage, hematide Chronic kidneydisease associated anemia, stomatitis, pharyngitis, diagnosticassessment of thyroid function, postmenopausal osteoporosis,hypercalcaemia, diagnosis of pancreatic exocrine dysfunction, andgastrinoma, Zollinger-Ellison syndrome, prevention of RDS in prematureinfants, and meconium aspiration syndrome, acute variceal bleeding,allergic rhinitis and conjunctivitis, spinocerebellardegeneration/ataxia, Short Bowel Syndrome, antibiotic, bactericidal,teriparatide is the only anabolic (i.e., bone growing) agent indicatedfor use in postmenopausal women with osteoporosis, Cortrosyn Analogue ofadrenocorticotrophic hormone (ACTH) used for diagnostic purposes,treatment of adrenal insufficiency, epilepsia, Chronic hepatitis B,chronic hepatitis C, primary and secondary immune deficiencies, acutedecompensated heart failure, colitis, esophageal variceal bleeding inpatients with cirrhotic liver disease and AIDS-related diarrhea,sarcoidosis and acute lung injury, and severe chronic pain; or whereinthe condition, disorder and/or disease is selected from erythropoieticporphyries, T2 diabetes, antifibrinolytic, central diabetes insipidus,delaying the birth in case of threat of premature birth, antibiotic,cystic fibrosis, angina, anticoagulant in patients with unstable anginaundergoing PTCA or PCI, systemic lupus erythematosus, hypercalcemia,osteoporosis, pagets disease, carbetocin works as an oxytocic,antihemorrhagic and uterotonic drug in the peripheral nervous system,prevention of uterine atony, induction, and control postpartum bleedingor hemorrhage, stimulant of the gastric secretion, for treathormone-sensitive cancers of the prostate and breast, inhibition ofpremature LH surges in women undergoing controlled ovarian stimulation,immunosuppression in organ transplantation to prevent rejection,peritumoral brain edema, diagnosis of ACTHdependent Cushing's syndrome,allergies, ankylosing spondylitis, psoriasis, chorioditis, erythema,keratitis, sclerosis, dermatomyositis, rheumatoid arthritis,Stevens-Johnson Syndrome, ulcerative colitis, diagnosis ofadrenocortical insufficiency, antibiotic, systemic infections caused bygram positive organisms, nocturnal enuresis, nocturia, and stoppage ofbleeding or hemorrhage in haemophilia A patients, acute hereditaryangioderma, postmenopausal osteoporosis, anti-parathyroid, Paget'sdisease, hypercalcaemia, hypertension, AIDS/HIV-1 infection, acutecoronary syndrome, unstable angina undergoing PCI, Alzheimer's andParkinson's disease, inhibition of premature LH surges in womenundergoing controlled ovarian hyperstimulation, Relapsing-RemittingMultiple Sclerosis, hepatic insufficiency, wound healing, inflammationof respiratory tract, asthenia, release of follicle-stimulating hormone(FSH) and luteinizing hormone (LH) from the anterior pituitary,stimulate the secretion of gonadotropin during disturbances fertility,and diagnosis of the functional capacity and response of thegonadotropes of the anterior pituitary, for skin lesions, surface woundsand eye infections, postmenopausal osteoporosis, Paget's disease,hypercalcaemia, hereditary angioedema, immune system related diseases,acromegaly, anticoagulant, fibroids and endometriosis, central diabetesinsipidus, Cushing's syndrome, diabetic foot ulcers, treatment ofcentral precocious puberty, uterine fibriods and endometriosis,vasodilatory, natriuretic, diuretic and neurohormonal effects,acromegaly, carcinoid syndrome, acute bacterial skin and skin structureinfections, initiation or improvement of uterine contractions, andcontrol postpartum bleeding or hemorrhage, hematide Chronic kidneydisease associated anemia, stomatitis, pharyngitis, diagnosticassessment of thyroid function, postmenopausal osteoporosis,hypercalcaemia, diagnosis of pancreatic exocrine dysfunction, andgastrinoma, Zollinger-Ellison syndrome, prevention of RDS in prematureinfants, and meconium aspiration syndrome, acute variceal bleeding,allergic rhinitis and conjunctivitis, spinocerebellardegeneration/ataxia, Short Bowel Syndrome, antibiotic, bactericidal,teriparatide is the only anabolic (i.e., bone growing) agent indicatedfor use in postmenopausal women with osteoporosis, Cortrosyn Analogue ofadrenocorticotrophic hormone (ACTH) used for diagnostic purposes,treatment of adrenal insufficiency, epilepsia, Chronic hepatitis B,chronic hepatitis C, primary and secondary immune deficiencies, acutedecompensated heart failure, colitis, esophageal variceal bleeding inpatients with cirrhotic liver disease and AIDS-related diarrhea,sarcoidosis and acute lung injury, and severe chronic pain.
 267. Themethod of claim 263, wherein the composition is coadministered with oneor more of an adjuvant, a chemotherapeutic agent, ananti-immunosuppressive agent, and an immuno stimulatory agent; whereinthe adjuvant is selected from the group consisting of CPG, polyIC,poly-ICLC, 1018 ISS, aluminum salts (for example, aluminum hydroxide,aluminum phosphate), Amplivax, BCG, CP-870,893, CpG7909, CyaA, dSLIM,Cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), IC30, IC31, Imiquimod,ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®,vector system, PLGA microparticles, imiquimod, resiquimod, gardiquimod,3M-052, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGFtrap, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, AsA404(DMXAA), 3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1dligands (such as C20:2, OCH, AH04-2, α-galatosylceramide,α-C-galatosylceramide, α-mannosylceramide, α-fructosylceramide,β-galatosylceramide, β-mannosylceramide), STING agonists (e.g. cyclicdinucleotides, including Cyclic [G(3′,5′)pA(3′,5′)p], Cyclic[G(2′,5′)pA(3′,5′)p], Cyclic [G(2′,5′)pA(2′,5′)p], Cyclic diadenylatemonophosphate, Cyclic diguanylate monophosphate), CL401, CL413, CL429,Flagellin, RC529, E6020, imidazoquinoline-based small molecule TLR-7/8a(including its lipidated analogues), virosomes, AS01, AS02, AS03, AS04,AS15, IC31, CAF01, ISCOM, Cytokines (such as GM-CSF, IL-2, IFN-a,Flt-3L), bacterial toxins (such as CT, and LT), any derivative of anadjuvant, and any combination of adjuvant; wherein the immunostimulatory agent is selected from the group consisting of anti-CTLA-4antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA,anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40,anti-GITR, anti-ICOS, anti-TIGIT, and inhibitors of IDO; wherein thechemotherapeutic agent is one or more of the following: aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide,filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron,hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan,lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate,metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole,ondansetron, paclitaxel, pilocarpine, prochloroperazine, rituximab,tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine,vincristine and vinorelbine tartrate.